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Members of the genus Bacillus are widely used in industry in the large-scale production of enzymes, such as proteases (4). Of particular industrial importance are proteases with activity at alkaline pH and high temperature. The best-known application for these alkaline proteases is their use in household detergents. Genes encoding alkaline protease have been cloned and sequenced from a variety of organisms, some of which are used commercially. To maximize industrial enzyme production, the host organism is generally manipulated to carry multiple copies of the gene. This can be accomplished by cloning the gene on a replicating plasmid, but preferentially this is achieved by amplification of the gene on the chromosome, as such amplification offers the more stable alternative. With an organism such as Bacillus subtilis, which can easily be transformed and has well-developed genetics, it is relatively straightforward to amplify genes on a chromosome; however, other Bacillus species frequently used in industry are more difficult to transform and less amenable to genetic manipulation. It is possible to use temperature-sensitive plasmids, such as pE194, as a means of achieving gene amplification on a chromosome; however, the incorporation of the plasmid origin of replication in the host chromosome is known to destabilize amplified structures (8, 13). There is therefore an interest in the development of improved amplification methods for industrial bacilli.

The alkalophilic species Bacillus lentus is an important industrial organism which produces a commercially important alkaline protease. In this report we describe the cloning and sequencing of the gene encoding an alkaline protease from the B. lentus isolate NCIB 10309 and the generation of a strain which contains multiple copies of this gene on the chromosome. In the process of generating such a strain we have developed a general method for gene amplification in poorly transformable bacteria.

Cloning of a B. lentus alkaline protease gene. Chromosomal DNA from B. lentus NCIB 10309 (6) was digested with the enzyme Sau3A, and fragments between 1.5 and 6.5 kb were purified and ligated into a general-purpose cloning vector, pSX50, developed at our laboratories (S. Hastrup, S. Branner, F. Norris, S. B. Petersen, L. Nørskov-Lauridsen, V. J. Jensen, and D. Aaslyng, 13 July 1989, International patent publication number WO89/06279), which had been digested with the enzyme BamHI. Ligated DNA was transformed into the protease-deficient mutant B. subtilis DN497 (S. T. Jørgensen, P. L. Jørgensen, and B. Diderichsen, 27 June 1991, International patent publication number WO91/09129) and transformants were selected on Luria-Bertani agar plates supplemented with chloramphenicol, 1% skim milk, and 0.2% xylose. Protease-producing transformants, which arose at a frequency of 10⁴, were identified by a clear halo around a colony. Two clones that carried the gene for subtilisin were isolated. The gene encodes a protein of 380 amino acids, consisting of a signal peptide, a propeptide, and a mature protein, in common with other alkaline proteases. The mature protein contains 269 amino acids and is currently marketed commercially under the trade name Savinase. A detailed description of its properties and its crystal structure have been presented elsewhere (2, 9, 11). The protein sequence has been deposited in the SwissProt database with the accession number P29600.

A new strategy for integration and gene amplification. To increase production of the protease, it was desirable to amplify the gene on the B. lentus chromosome. Amplified sequences on the B. subtilis chromosome have been shown to be destabilized by the presence of an active plasmid origin of replication in the integrated DNA (8, 13). We previously developed a strategy which avoids the presence of a functional plasmid replication system on the chromosome (7, 10). The amplification method is dependant upon the construction of a pUB110-derived plasmid incorporating two critically located plus origins of replication (+ori). Such plasmids have the capacity to form two separate progeny vectors, which we term the replicative and nonreplicative vectors, as described in the work of Tangney et al. (10). Each of the progeny vectors necessarily contains its own +ori, but by design only one of these progeny vectors (the replicative vector) encodes the trans-acting replication protein. As a consequence, the nonreplicative vector can be maintained only in a culture where either the replicative vector is also present (to supply the essential replication protein in trans) or it has integrated into the host chromosome and is replicated along with the chromosomal DNA. Therefore, by screening for loss of the replicative vector it is possible to identify clones where the nonreplicative vector DNA has integrated into the chromosome. In practice, the parental plasmid is designed so that the nonreplicative vector contains the DNA sequence with homology to the host chromosome that is the target for integration into the chromosome, by means of homologous recombination. As this vector cannot replicate autonomously, any subsequently derived amplified structures will not contain an active origin of plasmid replication.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Construction of the integrating plasmid pPL2002. Abbreviations are as follows: bla, the -lactamase gene; cat, the chloramphenicol acetyltransferase gene; +ori-pE194, the +ori of pE194; repF, the gene encoding the replication protein of pE194; repF, a deleted repF gene; erm, the erythromycin resistance gene; and S-309, the subtilisin gene from B. lentus NCIB 10309. Arrows indicate the directions of transcription.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   (A) Depiction of plasmids pPL2002 and pE194 when replicating in a Bacillus genetic background. The black rectangle represents a functional RepF protein (the product of the intact repF gene), which can act at both +ori pE194 sequences as indicated by the dashed lines. (B) Integration of pPL2002 into the B. lentus chromosome via the gene for subtilisin. Abbreviations are given in the legend to Fig. 1.

Amplification of the alkaline protease gene on the B. lentus chromosome. We tested the efficacy of the new approach by amplifying the alkaline protease gene of B. lentus. Plasmid pPL2002 was constructed as shown in Fig. 1. An MboI fragment of pE194, from positions 1 to 1585 (6), containing the +ori of pE194 and a segment of the gene encoding the replication protein RepF was cloned, in Escherichia coli, into pDN3000 (5) at the unique BglII site, resulting in plasmid pPL1957. Plasmid pSX120 contains a chloramphenicol resistance gene and the alkaline protease gene from B. lentus on a 3.3-kb EcoRI/BamHI fragment. This fragment was cloned into pPL1957 to generate plasmid pPL2002 (Fig. 1). This plasmid therefore contains the ampicillin resistance marker and functions for replication in E. coli; it also contains the cloned alkaline protease gene, a cat gene (which is functional in a Bacillus genetic background), the +ori from pE194, and a truncated repF gene.

Conclusions. We report the cloning, sequencing, and specific amplification of a protease gene on the chromosome of an alkalophilic bacillus. The strategy employed to generate the amplified strain is derived from a method which we previously developed using the plasmid pUB110 that precludes the presence of an active plasmid origin of replication on the amplified chromosome. In the new approach we adapted the earlier method for use with the temperature-sensitive plasmid pE194. The new strategy simplifies the plasmid constructions but perhaps even more significantly allows a direct screen for clones in which integration has occurred, by virtue of the temperature-sensitive nature of the plasmid. Strains in which integration has occurred are readily isolated by growth at the nonpermissive temperature in the presence of a selective antibiotic. Such strains can subsequently be used to derive clones where further gene amplification has occurred. While in the example presented here we detected spontaneous amplification of the alkaline protease gene on the B. lentus chromosome, it is also possible to screen for clones with multiple copies by successive subculturing of amplified strains in increasing concentrations of the relevant antibiotic, as described for other methods (10, 12).