Gene, 111 (1992) 125-130 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92]$05.00

125

GENE 06305

Cloning and characterization of a gene encoding extraceilular metalloprotease from Streptomyces lividans (Zinc-binding motif; LysR family; [t4C]u-casein assay; phosphoramidon; DNA-binding motif; 1,10-phenanthroline; secretion; signal sequence; plasmid plJ699)

Henri S. Liehenstein, LeighA. Busse, GregoryA. Smith, LindaO. Narhi, MichaelO. McGinley, MichaelF. Rohde, Jessica L. Katzowitz and Mark M. Zukowski Amgen Inc.. Thousand Oaks, CA 91320 (U.S.A.) Received by K.F. Chater: 23 July 1991 Revised/Accepted: 28 October/30 October 1991 Received at publishers: 27 November 1991

SUMMARY

The prt gene, encoding a protease (Prt) from Streptomyces lividans TK24, was cloned and sequenced. An S. lividans host with plasmid-borne pn secreted 200/Ag/ml of a 22-kDa Prt into the culture medium. Prt is classified as a metalloprotease since its activity is significantly inhibited by 1,10-phenanthroline or EDTA. The region upstream from prt codes for an incomplete open reading frame (ORF) oriented opposite to prt. This ORF has a strong similarity to a gene far,3ily(iysR) whose members regulate the transcription of structural genes required for either biosynthesis or degradation.

INTRODUCTION

There is a widespread interest in utilizing Gram + microorganisms such as Streptomyces spp. and Bacillus spp. for the secretion of eukaryotic proteins (Chang and Chang,

Correspondence to: Dr. H. Lichenstein, Amgen Inc., Thousand Oaks, CA 91320 (U.S.A.) Tel. (805)499-5725; Fax (805)498-8674.

Abbreviations: aa, amino acid(s); APMSF, (4-amidinophenyl)-methanesalfonyl fluoride; bp, base pair(s); BSA, bovine ~mm albumin; DFP~ diisopropyl fluorophosphonate; DMSO, dimethylsuifoxide;D'l'r, dithiothreitol; HPLC, high-performanceliquid chromatography; kb, kilobase(s) or 1000 bp; MES, 2-[N.raorpholino]ethanesulfonicacid; NBRF, National Biomedical Research Foundation; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF (Off), open reading frame(s); p, plasmid; PAGE, polyacrylamide-gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; Prt, protease; prt, gene encoding Prt; RBS, ribosome-bindingsite(s); SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; Th, thiostrepton; wt, wild type; [ ], denotes plasmid-carrier state.

1988; Lichenstein et al., 1988; Noack et ai., 1988; Keller et al., 1989; Mountain, 1989; Bender et al., 1990a,b). These bacteria efficiently secrete proteins directly into the medium and are easily cultivated on an industrial scale. However, one drawback of Gram ÷ secretion systems is that these organisms also secrete proteases that degrade the desired product (UImanen et al., 1985; Schein et al., 1986; Wong et al., 1986; Aretz et al., 1989). In Bacillus spp., this problem has been addressed by the development of protease-deficient strains (Kawamura and Doi, 1984; Stahlet al., 1984; Yang et al., 1984). These strains were constructed by cloning the genes for extracellular proteases, deleting essential coding regions of the genes in vitro, and replacing the wt chromosomal genes with the inactive counterparts. At least one of these protease-deficient strains was shown to improve the yield of a heterologous protein in Bacillus subtilis (Wong et al., 1986). There have not yet been any reports describing the construction ofa protease-deficient strain ofS. lividans. In fact,

126 numerous proteolytic activities have been identified in the culture medium from this strain (Aretz et al., 1~89), however, the genes coding for these proteases have not been isolated. As a first step toward the construction of a protease-deficient S. lividans, we cloned and sequenced a protease-specifying gene, prt, from this species. In addition, the prt gene product (PrO was purified and characterized.

(a) Construction and screening of a genomic library of Streptomyces lividans A S. fividans TK24 (Hopwood et al., 1983) genomic library was constructed by ligating partial Sau3AI fragments of TK24 chromosomal DNA into the BgiII site of pIJ699 (Kieser et al., 1988). TK24 protoplasts were transformed with the ligation mixture and plated onto R2YE plates (Hopwood et al., 1985). After 18 h, the plates were overlaid with a mixture (pre-warmed to 55°C) consisting of 90% skim milk (Difco), 0.4% agar, Img Th/ml (Sigma) and incubated at 30°C for an additional 2 days. A total of 2500 transformants were screened, and two colonies were found to be surrounded by zones of clearing (halos). These halos represent proteolytic breakdown of milk proteins and are indicative of enhanced protease secretion. Plasrnid DNA (pilL36) was isolated from the transforrnant with the larger halo and restriction analysis revealed that it contained a 9.0-kb insert. To subclone the putative prt, the 9.0-kb insert from pilL36 was purified as a HindIII fragment, partially digested with Sau 3AI and cloned into the BglII site of pIJ699. Using the screening procedure described above, we found that the smallest plasmid (pilL45) which allowed TK24 to produce halos contained a 3.4-kb insert. This 3.4-kb fragment was then cloned as a HindIII fragment into the E. coil vector pGEM7Zf( + ) (Promega, Madison, WI) to yield pGS4. A restriction map of the 3.4-kb insert is shown in Fig. 1. (b) Purification of Prt Fig. 2 shows the supernatant proteins of TK24[pHL45] compared to strain TK24[pIJ699]. At 36h, only the TK24[ pilL45 ] supernatant contained a detectable 22-kDa

i

I

I

I

I

2

3

94-67-43

14.4--Fig. 2. SDS-PAGE analysis of supernatant proteins in TK24[pHL45] and TK24[plJ699]. TK24[pHL45] and TK24[plJ699] were grown for 36h at 30°C in medium A (Lichenstein ctal., 1990) containing 5;tg Th/mL The culture supernatants were recovered by centrifugation and 5 pl of each supernatant was electrophoresed on a 12% Mini-Protean !! Ready Gel according to manufacturer's specifications (BioRad). The gel was stained with Coomassie Blue and destained according to published procedures (BioRad). Lanes: I, low-molecular-weight protein standards (Pharmacia): phosphorylasc b (94kDa)0 BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1kDa), u-lactalbumin (14.4kDa); 2, TK24[pHL45]; 3, TK24[plJ699].

protein (PRO. Prt was produced at approx. 200 #g]ml and had proteolytic activity (see section e). In order to localize the position of the prt gone in the 3.4-kb insert, we dotermined a partial aa sequence for Prt, designed a corresponding oligo to the sequence and hybridized restriction enzyme-digested pGS4 with this probe. Prt was purified from the TK24[pHL45] supern~tant by first diluting the broth from a 36 h culture with an equal volume of water and then centrifuging at 8000 x g. Supernatant was recovered and adjusted to 2.5 rnM EDTA/I mM PMSF, and immediately subjected to gel filtration chromatography on a Sephacryl S-300 column equilibrated in 20 mM MES/ 100rnM NaCI/2.5mM EDTA/I mM PMSF, pH 6.5. Fractions containing proteins were analyzed by SDS-

I I ~

prt

20.1--

I

I orl 1

1

30

EXPERIMENTAL AND DISCUSSION

}1 0

kDa

3,

4i ~

~"

Fig. 1. Restriction map of the 3.4-kb insert of pGS4. The arrows below the restriction map indicate the coding sequences for prt and orf/, Note that the complete sequence for off/is not contained in the insert (see section d).

127 PAGE and those fractions found to contain Prt were pooled and lyophilized. The powder was redissolved in 0.1% TFA and dia-filtered against 0.1% TFA to remove salt. The TFA was then evaporated and the pure protein was stored as a powder at -20°C until further use.

has features which suggest that Prt is initiaJ~y synthesized with a signal sequence. Because the N terminus of the mature Prt found in the culture medium was blocked, we could not accurately determine a signal peptide cleavage site, nor could we determine whether Prt also has a propeptide as described in other Streptomyces proteins (Robbins et al., 1984; Henderson et al., 1987; Nakai et al., 1988). Based on the deduced aa sequence, full length Prt is expected to consist of 227 aa with a calculated Mr of 23 765. The aa sequence of Prt was compared to sequences in the NBRF data base (Release 27.0, 12/90) and the Swiss Prorein data base (Release 17.0, 2/91) using the UWGCG WordSearch and FastA programs. No protein in either data base showed significant similarity to Prt. An interesting feature of the Prt aa sequence is a region (aa 160-170) that corresponds to a Zn 2+-binding motif (Jongenoel et al., 1989) which can be found in a number of Zn2+-dependent metalloproteases (Table I). In thermolysin, e.g., it is known that two His residues in this motif are zinc ligands (Matthews et al., 1972). Analogous His 163and His ~67 residues in Prt may serve as zinc ligands, but further experimental evidence is required. The coding sequence of prt is characteristic of many Streptomyces genes in that it has a high overall G + C content (71.8%) and a strong tendency (96.0%) to utilize codons that have G or C in the third position (Hopwood et al., 1986). As in many Streptomyces genes, there is also a perfect inverted repeat sequence following the stop codon which could serve to terminate transcription of prt. Upstream from prt is another ORF (Figs. I and 3) which also has the typical codon usage of Streptomyces genes. The off1 gone is oriented opposite to prt and most likely uses GTG as an initiator codon since this codon is preceded by a putative $treptomyces RBS. Plasmid pGS4 does not contain a full-length gene encoding Orfl, thus Offl has a minimum of 302 aa. The UWGCG FastA program was used to search for proteins similar to Orfl in the Swiss Protein data base. Proteins with the strongest similarity to Orfl all belong to

(c) Partial aa sequence of Prt and design of corresponding oligo An attempt to determine the N-terminal aa sequence of purified Prt revealed that it was blocked. To determine internal aa sequences, purified Prt was digested with sequencing grade h-ypsin (Boehringer Mannheim) for 18 h at 37 ° C. Resulting peptides were separated on a Vydac C4 HPLC column previously equilibrated in 0.1% TFA in water. Peptide fragments were eluted using a linear gradient up to 68% acetonitril¢ in the same buffer. Fractions with high absorbances at 280 nm were dried and run on an Applied Biosystems 477 protein sequencer. The aa sequence of one tryptic fragment was determined to be: L-A-E-S-S-S-G-A-D-F-A-Y-Y-E-G-N-D-S-R. Based on codon preferences in Streptomyces (Hopwood et al., 1986), a mixed 33-met oligo corresponding to the underlined aa was synthesized as follows: 5 ' - G G c~ GCcG GAT TTC GCg TAC TAC GAG G G ~ AAC GAC. (d) Nucleotide sequence and analysis of prt from

Streptomyces lividans Intense hybridization of the mixed 33-mer oligo to specific restriction fragments of pGS4 confirmed that the recombinant plasmid carried prt. The nt sequence of prt including 5' and 3' flanking sequences was determined (Fig. 3). An ORF coding for Prt was localized between nt 1147 and 1827. The deduced aa sequence included the tryptic fragment described above as well as other trypd-,. peptide fragments that were sequenced (data not shown). A putative RBS which showed complementarity to the 3' end of the 16S ribosomal RNA of S. lividans (~[t~b and Cohen, 1982) was found to precede the start codon at position 1147. The N terminus of the deduced aa sequence TABLE I

Comparison of putative Zn 2 ~-binding sequences in metalloproteases of bacterial orion Protease

Sequence a

Streptomyces lividans Prt Streptomyces cacoi Npr Bacillus thermoproteolyticus Thermolysin Bacillus subtilis NprE Serratia sp. protease Pseudomonas aeruginosa LasB Legionella pneumophila PepI Erwinia chyrsanthemi ProB

T T V T F A G F

A V A A T A G T

H H H H H H H H

Reference E E E E E E E E

T A L M 1 V V I

G G T T

G S S

H V H S H A H G H A H G tH G

L L V V L F F L

This paper Chang et al. (1990) Titani et al. (1972) Yang et al. (1984) Nakahama et al. (1986) Bever and Iglewski (1988) Black et al. (1990) Delepelaire and Wandersman (1989)

a The sequences were aligned as described (Jongeneel et al., 1989) and the three invariant residues in the putative Zn: +-bin~ing motif ~L'eboxed.

128 aagcttctagaGATCCCGCCCCCTTCCAGCCACTCCCGGTACGCGGTGGACTGGC~GCG I G G G E L N E R Y A T S Q R A

60

GCCTCC~GTACGCGTCCTC~GGGCGGGG~GACCGCGTCCAGCTCGGCCCGGGTGCGG A E W Y A D E L A P F V A D L E A R T R

120

GCGGCCAGCAGCAGGCGTACGCCGAGGGGGTCGCCGTACAGGCGCCGCACCGCCGTGTCG A A L L L R D G L P D G Y L R R V A T D

180

G~CGG~CTGGG~CTGGGCTGGCAGACCGTGACGACCTCGCCGGTGGCGACCAGGGAG P R S n S S P Q C V T V V E G T A V L S

240

GCGGCCGTGTGGTAGTCGCCGTGCAGCACCCGGGGGTTGAGGCCGACGGCGCGCAGCATG A A T H Y D G H L V R P N L G V A R L M

300

C~TGCACGCCGTCCCATTCGCCGTCGACGGTGGGGTCGACCATC~CCGGTCGTCGGCG R H V G D W E G D V T g D V M W R D D A

360

AGGTCGGCGAGGCGGACGACGGGCCGGGCGGCCGCGGGGTGGTCGGCGGGCAGCATGACG L D A L R V V P R A A A P H D A P L M V

420

~CTGCGGTTCGCGCTCGACCAGGACGCGCAGCCGCAGGTCCTCGGGGATGTGCAGCGCA F Q P E R E V L V R L B L D E P I H L A

480

CAGCCCTCGACCTCGTGCACG~GGCGACGTCGAGCTGACCGTCGGTCACCCGGCGCAGC C G E V E H V F A V D L Q G D T V R R L

540

AGCGCGTTGGCGGAGACGTCCATCTGCAGGGTCGGTTCCAGGCCCGGCCGTCTGAGCCGG L A N A S V D M Q L T P E L G P R R L R

600

CGCAGCCAGCCGGCCAGGGCCCGGCTGGCCGTGGAGCCGACCCGCAGCCTGCTGTCGCCG R L W G A L A R S A T S G V B L R S D G

660

CCGGCCGCGGCGGCGCGGGCCTCGCGGACCAGGGTGTTCATGTCGGTCAGCAGCGGACGG G A A A A R A E R V L T N M D T L L P R

720

GCCCGGCCGAGCACCGTCCGGCCGAGCGGGGTGGGGCGGCAGCCGGTGCGCTCCCGGGTG A R G L V T H G L P T P R C G T R E R T

780

~CAGCGGTCCGCCCAGGGCCTGTTCGATGCGGGTC~CTGAGTGCTC~CGTGGGCTGT F L P G G L A Q E I R T L Q T S L T P Q

840

GCGACACCCAGTCGGCGTGCCGCCCGGTGCAGGCTGCCGGCGTCGGCGATGGCGCACAGT A V G L R R A A R H L S G A D A I A C L

900

GCGCGTAGGTGCCTCACCTCGAGCTCCATGCAGGGAGCGTAAAGCGG~CAGTTGGTTGC A R L H R V

960

GCCAGGTG~CAAAACGCGGCGGATCAGGGCGAGTTCTGCACTCTGGTCAAAGCTGG~C

1020

287 267 247 227 207 187 167 147 127 107 87 67 47 27 7 1

GAGAGTGGCCGGGCGGTGGGTGATAGCCCGGCCCTATCACCTGTTGCCATCATCACAGCG 1080 GGCTCATGGGCGCCCCACACTCACCGGTGACGACTTCTCCCCACTCCCCCA~C~GGAG 1140 ~rt~ M R I T L P L L S T A V G L G L T A 18 TC~TCGATGCGTATCACCCTGCCCCTTCTTTCCACCGCGGTCGGTCTCGGCCTGACGGCC 1200 A V L G T G P A A T A A A P Q E P V R A 38 GCCGTGCTCGGCACCGGCCCCGCCGCGACGGCCGCGGCGCCCCAGGAGCCGGTCAGAGCC 1260 A Q L G Y Q P S A G S G E D A A A N R A 58 GCCCAGCTCGGCTACCAGCCCTCGGCCGGCTCGGGCGAGGACGCGGCCGCC~CCGCGCG 1320 F F E A V V K S V A E K R A A N P S A A TTCTTCGAGGCGGTCGTC~GTCCGTCGCCGAG~GCGCGCCGCC~CCCGTCCGCCGCC

the LysR family of transcriptional regulators (Henikoff et ai., 1988). There is particularly strong similarity between aa 18-37 in Orfl and comparable regions in LysR proteins. This region has been postulated to represent a helix-turnhelix DNA-binding domain (Pabo and Sauer, 1984: Dodd and Egan, 1990). A preliminary experiment suggested that Orfl plays a role in prt expression in spite of missing an undetermined number of authentic C-terminal aa. In this experiment, pHLA5 was digested with Pstl and religated, thereby resulting in the removal of a 1.0-kb PstI fragment. The resultant plasmid, pGS6, is thus deleted off the Cterminal 184 aa in Orfl. When pGS6 was transformed into TK24, no protease production was observed, suggesting that Orfl is necessary for prt expression.

78 1380

A A V T V Y Y S A T N A P S F R S Q I S 98 GCGGCCGTCACCGTCTACTACAGCGCCACC~CGCGCCGAGCTTCCGTTCCCAGATATCC 1440 R S A Q I W N S S V S N V R L A E S S S 118 CGCTCCGCCCAGATCTGG~CAGCTCGGTGTCC~CGTACGGCTCGCGGAGTCGAGTTCC 1500 G A D F A Y Y E G N D S R G S Y A S T D 138 GGCGCGGACTTCGCGTACTACGAGGGC~CGACTCGCGCGGCTCGTACGCGTCCACGGAC 1560 G H G S G Y I F L D Y R Q N Q Q Y D S T 158 GGGCACGGCAGCGGCTACATCTTCCTCGACTACCGCCAGAACCAGCAGTACGACTCGACC 1620 [{ V T A H E T G H V L G L P D H Y S G P 178 CGCGTGACCGCCCACGAGACCGGGCACGTGCTCGGCCTGCCCGACCACTACTCCGGGCCG 1680 C S E L M S G G G P G P S C T N P Y P N 198 TGCAGCGAGCTGATGTCGGGCGGCGGCCCCGGCCCGTCCTGCACC~CCCCTACCCG~C 1740

(e) Characterization of Prt proteolytie activity Prt was purified from TK24[pHL45] broth by gel filtration chromatography on a Superdex 75 column using the Pharmacia FPLC system and a stabilizing buffer consisting of 34~o sucrose/2~o dextrin/0.5% Na2HPO4/0.1~o KH2PO4/0.1% MGSO4/0.5% NaCi, pH 7.0. Fractions containing protein were analyzed by SDS-PAGE, and those fractions containing purified 22-kDa Prt were pooled and assayed. The pH optimum of Prt was determined to be 7.7 using an azocoll assay (Table lI). The temperature optimum of Prt, as determined by activity toward [14C]~casein, was found to be 50°C, with the enzyme retaining some activity even at 70°C (Table Ill). To verify that Prt is a metalloprotease, the effect of several different inhibitors on the activity of purified Prt was determined (Table IV). All serine protease inhibitors tested had no eff~t on Prt activity. As expected for a metalloprotease, the chelating agents, EDTA and 1,10-phenanthroline completely inhibited Prt activity. Prt activity could be restored upon the addition of ZnCI2 to the EDTA-treated enzyme or to purified Prt itself, thus providing fu~her support that Prt is a Zn 2+-dependent metalloprotease. Interestingly, the metalloproteasc inhibitor phosphoramidon had no effect on Prt activity. PhorphoramJdon is

S T E R S B V N Q L W A Y G F Q A A L D 218 TCCACCGAGCGCAGCCGGGTG~CCAGCTGTGGGCCTACGGCTTCCAGGCCGCCCTCGAC 1800

TABLE II

K A L E K A S Q R , 227 ~GGCGCTGGAG~GGCCTCCCAGCGCTGACGTACGCGGACCACCGTGCGGGCGGCCCGG 1860

pH optimum of Prt

)

CCGGGCCGCCCGCACGCGTGCGCGCTCCCTCCACCTCCGCTTGGGC~CC