Vol. 57, No. 4

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1991, p. 901-909

0099-2240/91/040901-09$02.00/0 Copyright C) 1991, American Society for Microbiology

Cloning, Characterization, and Multiple Chromosomal Integration of a Bacillus Alkaline Protease Gene JOOP C. VAN DER LAAN,* GIJS GERRITSE, LEO J. S. M. MULLENERS, RUUD A. C. VAN DER HOEK, AND WIM J. QUAX

Royal Gist-brocades N.V., Research and Development, P.

0.

Box 1, 2600 MA Delft, The Netherlands

Received 20 August 1990/Accepted 9 January 1991

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.

A wide variety of Bacillus species (24) secrete serine endoproteases into the external medium. Bacillus serine proteases have their best-known application in detergent powders. To best meet the alkaline conditions in detergents, serine proteases with a highly alkaline pH optimum (referred to herein as high-alkaline proteases) are preferred above the subtilisins that have an optimal pH of 8.5 to 10. After a screening program, Zuidweg et al. (47) isolated an alkalophilic Bacillus strain (PB92) that produced a high-alkaline serine protease (PB92 protease) with unique pH optimum of 10.5 to 12. The PB92 protease performed extremely well in detergents. Amino acid sequences for the serine proteases from Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis have been determined (23, 26, 34), and the genes from B. amyloliquefaciens, B. licheniformis, B. subtilis, B. subtilis subsp. amylosacchariticus, and the alkalophilic Bacillus sp. strain YaB have been cloned and sequenced (13, 17, 36, 38, 41, 45). The complete amino acid sequences of thermitase (25) and proteinase K (15) have also been reported. These two enzymes are related to subtilisins but differ from them in containing cysteine(s). The DNA sequences of the cloned subtilisins reveal an intervening propeptide sequence between a signal sequence and the mature enzyme (13, 17, 36, 38, 41, 45). It was shown (27) by analysis of subcellular fractions of Bacillus strains expressing various mutations in the subtilisin gene that the proprotein of subtilisin exists in association with the cell membrane. Furthermore, the conversion of the primary gene *

product into the mature enzyme is mediated by active subtilisin, and therefore this processing is most likely autocatalytic (27). The function of the prosequence has not been well established. It is structurally well conserved among the various subtilisins and is highly enriched with charged residues. It is speculated that these residues are important for interaction of the prosequence with the mature portion of subtilisin, which is most likely essential for folding of subtilisin into the enzymatic active conformation (11). To overproduce a protease, two strategies for increasing the gene copy number in Bacillus can be considered: (i) using plasmid-containing strains and (ii) using strains containing additional genes integrated in the chromosome. In B. subtilis, replicative plasmids harboring cloned sequences can be prone to either segregational or structural instability (4, 9, 21, 28). Chromosomal integration has been applied to solve these problems. Although these strains are more stable than plasmid-containing strains, instability of tandemly amplified chromosomal sequences has also been reported (1, 44). For improved production of the PB92 protease, we developed a transformation and chromosomal integration system for Bacillus alcalophilus. MATERIALS AND METHODS Bacterial strains and plasmids. The alkalophilic strain B. alcalophilus PB92 (ATCC 31408) was isolated from the soil,

and the gene that encodes the high-alkaline protease was cloned. B. subtilis 1-A40 (43), which has a relatively low extracellular protease activity, was used as the host for cloning. pUB110 (8) was used as a cloning vector in B.

Corresponding author. 901

902

VAN DER LAAN ET AL.

subtilis. Plasmid pMAX-4, a derivative of pE194 (12) carrying the neomycin resistance (Kmr) gene of pUB110 and the alkaline protease gene of strain PB92, was used as an integration vector to construct B. alcalophilus strains PBT108 and PBT109. Integrants were constructed by using PB92', a classical mutant strain of PB92 with increased protease production, as the starting strain. Media. All Bacillus strains were grown in tryptic soy broth (TSB; Oxoid) aerobically at 37°C. This medium, which contains 20 ,ug of neomycin per ml, was used to isolate plasmid DNA. For the isolation of transformants containing the PB92 protease, minimal medium (2) containing 50 Kg of tryptophan per ml, 20 Kg of methionine per ml, 20 ,ug of lysine per ml, and 20 ,ug of neomycin per ml was used. Recombinant DNA techniques. Chromosomal DNA from B. alcalophilus PB92 was obtained by the method of Saito and Miura (31). Plasmid DNA was isolated by a modification of the method Birnboim and Doly (3) in which lysozyme incubation was performed for 5 min at 37°C. Restriction endonucleases and other enzymes were purchased from GIBCO-BRL and used according to the instructions of the supplier. Transformation of B. subtilis was performed by the method of Anagnostopoulos and Spizizen (2). DNA fragments from restriction enzyme digests were resolved and analyzed by electrophoresis on 0.8% agarose gels (10). For hybridization experiments, the separated fragments were transferred to nitrocellulose by the method of Southern (35) and detected with DNA probes labeled by nick translation

(29).

For cloning of the PB92 protease, cells from the transformation mixture were plated on minimal plates containing 2.8% K2HPO4, 1.2% KH2PO4, 0.4% (NH4)2SO4, 0.2% trisodium citrate 2H20, 0.04% MgSO4 7H20, 0.00005% MnSO4 4H20, 0.4% L-glutamic acid, 0.5% glucose, 0.02% Casamino Acids, 50 ,ug of tryptophan per ml, 20 ,ug of methionine per ml, 20 Fg of lysine per ml, 20 ,ug of neomycin per ml, 0.4% casein, and 1.5% agar. After overnight incubation, the increase in protease production was determined by observing the precipitation of halos of casein cleavage products around colonies in the agar plates. DNA sequencing. Specific restriction fragments of pM58 were cloned on the appropriate M13 vector (mplO, mpll, or mpl8) and sequenced by the chain terminating dideoxy method (32). Transformation. The polyethylene glycol-induced protoplast transformation method of Chang and Cohen (5) with the following modifications was used to transform the alkalophilic strain PB92'. (i) Protoplasts were prepared in alkaline holding medium containing 0.5 M sucrose, 0.02 M MgCl2, and 0.02 M Tris-maleate buffer (pH 8.0) to which 0.4 mg of lysozyme per ml was added. (ii) The protoplasts were pelleted and suspended in 5 ml of alkaline holding medium to which 3.5% (wt/vol) Bacto-Penassay broth and 0.04% albumin merieux were added. The transformed protoplasts were regenerated on modified DM3 (5) plates containing 8.0 g of Gelrite Gellam Gum (Kelco), 0.3 g of CaCl2 2H20, 4.06 g of MgCl2 H20, 5.0 g of N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (Sigma), 5.0 g of Casamino Acids, 5 g of yeast extract, 1.5 ml of 4 M NaOH dissolved in 750 ml of H20 and mixed after sterilization with 250 ml of 2 M sucrose and 10 ml of 50% (wt/vol) glucose, 1 ml of albumin merieux, 10 mg of thiamine, 5 mg of biotin, and 50 mg of neomycin. Plates were poured at approximately 70°C. Transformation of competent B. subtilis cells was performed as described previously (37). Preparation of competent cells and transformation of Escherichia coli were described pre-

APPL. ENVIRON. MICROBIOL.

viously (19). Chromosomal DNA was prepared by the method of Saito and Miura (31). Isolation of chromosomal integrants. After transformation with integration plasmid pMAX-4 (see Fig. 6), the transformant was inoculated in TSB containing 1 or 20 ,ug of neomycin per ml and incubated for 24 h at 37°C. A 5-ml sample of the culture obtained was diluted in 100 ml of the same medium and incubated for 24 h at 50°C. The last procedure was repeated twice. The cell culture was plated on heart infusion plates containing 1 or 20 Fg of neomycin per ml and incubated for 24 h at 50°C. Chromosomal DNA of potential integrants was isolated (31) and characterized by restriction enzyme analysis and Southern blotting experiments. Protease production. B. alcalophilus PB92-derived strains were fermented in 10-liter Eschweiler fermentors in a medium containing 22 g (based on dry matter) of yeast per liter, 5 g of K2HPO4 3H20 per liter, 0.05 g of MgSO4 * 7H20 per liter, 0.05 g of CaCl2 per liter, 0.005 g of FeSO4 7H20 per liter, and 0.05 g of MnSO4 . 4H20 per liter. The medium components were dissolved in 90% of the final volume and sterilized at pH 7.0 at a temperature of 120°C for 1 h. The inoculation culture was obtained by inoculation with a PB92-derived strain into 100 ml of TSB; after sterilization, 4 ml of 1 M sodium carbonate solution was added from a slant tube. The inoculation culture was incubated at 37°C for 24 h on a shaking apparatus. The medium was inoculated at 37°C and a pH of 8.0 with 1 volume of the inoculation culture per 100 volumes of medium. The main fermentation was carried out at 37°C in stirred fermentors equipped with devices to control pH, temperature, and foaming and a device for continuous measurement of the dissolved oxygen concentration and the oxygen uptake rate. At 17 h after inoculation, a -

pUB 110 (KmR; 4.5 kb)

Bacillus PB92DNA

cut BamHI

cut partial Sau3A

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Selection

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1-A40

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FIG. 1. Cloning of the high-alkaline protease gene of the alkalophilic strain B. alcalophilus PB92.

BACILLUS ALKALINE PROTEASE GENE

VOL. 57, 1991 P1. 20

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FIG. 2. DNA and deduced amino acid sequence of the alkaline protease of B. allcalophiliis PB92. Both strands have been sequenced from several independent overlapping clones. The putative ribosome-binding site (RBS), two putative promoters (P1, P2), and the transcriptional terminator (T) are underlined. The NH2 terminus of the mature protease was checked and confirmed by NH,-terminal amino acid sequencing.

30% glucose solution sterilized at 120°C for 1 h was added to a final concentration of 30 g of glucose per liter of medium. Protease activity. Protease activity was assayed with dimethylcasein as a substrate as described by Lin et al. (20). RESULTS of a high-alkaline serine protease of the alkalophilic Cloning strain B. alcalophilus PB92. Chromosomal DNA of strain PB92 was partially digested with Sau3A and ligated into BamHI-digested pUB110. B. subtilis 1-A40 was transformed with the ligated mixture and plated on minimal plates containing 0.4% casein. After 17 h, one colony of the neomycinresistant transformants was found to form a large protease halo. A recombinant plasmid containing a 1.8-kb DNA insert

was purified from the halo-forming transformant and designated pM58 (Fig. 1). Southern blot DNA-DNA hybridization of several chromosomal DNA digests of strain PB92 with 32P-labeled pM58 as a probe showed the positive bands expected from the restriction pattern of pM58 (data not shown). B. subtilis 1-A40 cells harboring pM58 showed a 60-fold increase of extracellular protease production compared with that of control B. subtilis 1-A40 cells harboring plasmid pUB110. The protease produced was sensitive to inhibition with phenylmethylsulfonyl fluoride but resistant to EDTA, indicating that a serine protease had been cloned. Nucleotide sequence of the alkaline protease from strain PB92. The nucleotide sequence of the cloned protease gene and its flanking DNA regions was determined (Fig. 2). There is an open reading frame between nucleotides 115 and 1254

APPL. ENVIRON. MICROBIOL.

VAN DER LAAN ET AL.

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that encodes a polypeptide of 380 amino acids consisting of three parts: (i) a signal peptide of 27 amino acids containing positively charged amino acids close to its N terminus, a core of uncharged amino acids, and a putative cleavage site (for a review, see reference 18); (ii) an intervening propeptide of 84 amino acids that is cleaved off during maturation, as known for other proteases (13, 17, 36, 38, 41, 45); and (iii) a mature protease of 269 amino acids. The open reading frame is preceded by a Shine-Dalgarno sequence (33); based on the B. subtilis consensus promoter sequence (16), two putative promoters (P1, P2) can be Pro-sequences

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identified. It is followed by a sequence which might form a hairpin structure and a cluster of T's, i.e., the structure typical for a rho-independent transcription terminator (30). Comparison of the amino acid sequence of strain PB92 alkaline protease with other serine proteases. The amino acid sequence of the mature PB92 protease shows homology with other serine proteases: subtilisin BPN' (23, 41), subtilisin DY (26), subtilisin Carlsberg (13, 34), protease YaB (17), and thermitase (25) (Fig. 3). The homology with protease YaB (82%), also produced by an alkalophilic organism, is high compared with the homology shown with subtilisins (60 to 61%) (Fig. 4). The lowest homology is found with thermitase produced by Thermoactinomyces vulgaris (46%). In both PB92 protease and protease YaB there are six residues deleted in comparison to subtilisin BPN', with a notable deletion of four amino acids around position 160. Comparative studies involving the prosequences of the enzymes produced by bacilli were performed (Fig. 4 and 5). All prosequences are enriched with charged residues, but a striking difference is seen between the prosequences of the enzymes produced by alkalophilic bacilli and those produced by neutrophilic bacilli. The first group mainly contains an excess of negatively charged residues, whereas the second group has a random distribution of both negatively and positively charged residues. This feature is reflected in the net charges of the prosequences in question: PB92 protease

BACILLUS ALKALINE PROTEASE GENE

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