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Applied and Environmental Microbiology, October 2004, p. 6337-6341, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6337-6341.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning and Expression of clt Genes Encoding Milk-Clotting Proteases from Myxococcus xanthus 422

M. Poza,1 M. Prieto-Alcedo,1 C. Sieiro,2 and T. G. Villa1*

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela,1 Department of Microbiology, Faculty of Sciences, University of Vigo, Vigo, Spain2

Received 29 January 2004/ Accepted 20 June 2004


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ABSTRACT
 
The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.


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INTRODUCTION
 
Currently, there are three main types of rennins used by the cheese-making industry: (i) rennins extracted from the abomasum of suckling ruminants (such as chymosin [EC 3.4.23.4]), (ii) rennins prepared from microbial broths, and (iii) recombinant rennins. In general, animal rennins are not sufficient to cover world demands, and this fact has prompted research into both microbial and recombinant rennins. Although a variety of bacteria, yeasts, and fungi have been isolated as natural producers of milk-coagulating enzymes (3, 10, 13, 26, 32, 45), only two genera are used worldwide in cheese production: Mucor (12, 39) and Endothia (8, 15). Several aspartic protease genes from molds and bacteria have been cloned and expressed (15, 22, 33, 44). Bovine chymosin has been cloned and successfully expressed in Escherichia coli (11, 18, 25, 27), in Bacillus spp. (19, 28), in yeasts (14, 24, 38, 42), and in filamentous fungi (7, 40, 41). In 1988, a recombinant bovine chymosin expressed in E. coli was first introduced into the market with the final aim of gradually replacing animal rennins (16, 20). Some studies have shown that Myxococcus xanthus is able to biosynthesize and secrete large amounts of extracellular molecules of biotechnological potential (6, 9), including proteases of interest to the cheese-making industry (4, 17, 29, 30, 34). The present work describes the isolation from M. xanthus 422 of genes able to induce the milk-clotting phenotype when E. coli, Saccharomyces cerevisiae and Pichia pastoris expression systems are used.

First, a genomic library of M. xanthus 422 (Spanish Type Culture Collection) was constructed in E. coli XL1-Blue. The myxobacterial strain was grown in casitone tryptone medium (30), and its genomic DNA was extracted with a Wizard genomic purification kit (Promega). Then, DNA was partially digested with the enzyme Sau3A1, and the fragments obtained were ligated to the pUC18 vector, previously digested with BamHI.

The total number of E. coli XL1-Blue clones harboring 2- to 6-kbp inserts (average size, 4 kbp) was estimated to be 20,000, with a 0.999 probability of having any given gene represented (5). White E. coli colonies grown on Luria-Bertani (LB) plates prepared with ampicillin, IPTG (isopropyl-ß-D-thiogalactopyranoside), and X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), as described previously by Sambrook et al. (35), were screened for milk-clotting ability by a modification of the method proposed by Emtage et al. (11). Each recombinant colony was grown for 24 h at 37°C and with shaking at 100 rpm on microtiter plates containing 125 µl of LB medium with ampicillin (50 µg/ml) and 0.4 mM IPTG (46). In order to evaluate intracellular activity, pelleted cells were lysed by adding 25 µl of a solution containing 10 mg of lysozyme per ml in 10 mM Tris-HCl, pH 8. Lysis was maintained for 1 h at 37°C, after which 0.1 M HCl was added to bring the pH down to 2. After 5 min, the pH was raised to 6.3 by the addition of 0.1 M NaOH. Finally, the plates were kept at room temperature for 1 h.

A volume of 200 µl of substrate (26% [wt/vol] skim powdered milk diluted in 10 mM potassium phosphate buffer, pH 6) was added both to the lysed cellular debris and to 50 µl of each culture supernatant obtained. All plates were incubated at 37°C with gentle stirring until milk clots became apparent; some of the clones were readily detectable since, after being inverted, the wells remained filled (Fig. 1). The colonies exhibiting the phenotype were cultured in increasing broth volumes to ascertain their activity, and eight clones that showed the unambiguous property of forming milk clots were finally obtained. In all cases, milk-clotting activity remained intracellularly located.



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  		FIG. 1. Screening of the library by detection of milk-clotting activity on microtiter plates. Left, arrows indicating milk clots developed by two positive clones; right, detail of a well filled with a milk clot.

DNA sequencing of the inserts contained in the eight positive clones was performed according to the method of Sanger et al. (36), by employing duplex DNA and aT7 Sequenase Quick-Denature plasmid sequencing kit (Amersham Pharmacia Biotech) and taking into account the high G+C content of myxobacterial DNA. Labeling was carried out with Redivue [35S]dATP. Sequence initialization was accomplished with the universal primers m13 and m13rev, and oligonucleotides were later designed as the sequence proceeded.

The sequences were assembled and analyzed using both DNAstar (Lasergene) and Vector NTI (Informax) software and the BLAST 2.0 application (1) available on the Web site of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). A total of 26.6 kbp was deposited in GenBank. Following this, it was possible to locate a total of 26 putative open reading frames (ORFs) (Table 1) by use of the software mentioned above. The high percentage of G+C, as well as codon usage, showed that the coding sequences that preferentially possess G or C at the third position of the codon were typical of myxobacteria (23).


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  		TABLE 1. Twenty-six putative ORFs found in eight milk-clotting clones obtained from the M. xanthus 422 library screening

As shown in Table 1, clone 8 contained two ORFs (USC8-1p and USC8-2p), separated by three nucleotides, and they exhibited a high degree of similarity (99.6 and 98.8%, respectively) to PrtB and PrtA, as described by Quillet et al. (31). According to those authors, both genes are linked in the same operon, forming part of the same transcriptional unit, the prtA gene coding for a serine protease and the prtB gene encoding a zinc metalloprotease.

The next step consisted of subcloning the ORFs with the potential to cause the milk-clotting activity (see boldface ORFs in Table 1). The choice of such ORFs was based on their size and the degree of similarity with corresponding proteins from the databases, but first all of those showing a certain degree of similarity to corresponding proteins not related to proteases were discarded.

The ORFs selected were PCR amplified by using the oligonucleotides shown in Table 2 and a mixture of Taq and Pfu polymerases (7:3) to ensure blunt end formation. The annealing temperature or elongation time was modified as needed, depending on the length of the DNA fragment or on its G+C content, although the general cycling program was 1 cycle at 94°C for 7 min and 25 cycles of 94°C for 1 min, 50°C for 2 min, and 72°C for 7 min. The process was completed with a final cycle of 72°C for 7 min.


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  		TABLE 2. ORFs selected for subcloning and oligonucleotides used for their PCR amplification

The PCR products obtained were cloned in a pCR-Blunt II-TOPO vector (Invitrogen) by use of a Zero Blunt TOPO PCR cloning kit (Invitrogen) and the E. coli TOP10 strain (Invitrogen) for transformation techniques, following the manufacturer's instructions. It should be noted that a joint subcloning of ORFs USC8-1p and USC8-2p was accomplished, since Quillet et al. (31) described that the deletion of either the prtB or prtA gene leads to a loss of activity.

These new constructions subsequently allowed us to easily carry out subclonings of the ORFs in the expression vectors described below.

Next, the ORFs cloned in the pCR-Blunt II-TOPO vector were extracted by EcoRI digestion, thus ensuring the presence of cohesive ends, and ligated to an EcoRI-digested pUC18 vector (Amersham Pharmacia Biotech) in order to analyze their expression in E. coli XL1-Blue, after separation from the rest of the genetic material contained in the eight original clones. The recombinant E. coli XL1-Blue colonies were selected in LB medium with ampicillin (50 µg/ml), IPTG, and X-Gal (35). The orientation of the inserts with respect to the lac promoter was analyzed, and clones containing the ORFs inserted appropriately were selected. Recombinant colonies were cultured using 0.4 mM IPTG as the inducer of the lac promoter.

Concentrated extracellular supernatants, as well as intracellular extracts, were prepared (30, 43) and subjected to milk-clotting assays in which 1 ml of substrate (26% [wt/vol] skim powdered milk diluted in 10 mM KH2PO4, pH 6) was mixed with 1 ml of each protein sample and incubated at 37°C until milk clots were visualized.

Only two recombinant E. coli strains, one containing ORF USC3-4p and the other containing USC8-1p and USC8-2p cloned together, showed milk-clotting activity, although in both cases the enzyme remained intracellularly located, probably due to the lack of functional secretion signals. Another cloning strategy, such as a fusion protein construction, could be used for improving the secretion of products encoded by M. xanthus DNA in E. coli.

ORFs USC8-1p, USC8-2p, and USC3-4p were assigned the function of milk clotting and termed, respectively, cltA (synonym, prtB), cltB (synonym, prtA), and cltC (which do not show any significant similarity with other known protease genes) and GenBank submissions, including the new names, were updated (Table 1).

Next, the cltA, cltB, and cltC genes were ligated to the pYES2 vector (Invitrogen), which had been previously digested, and these new constructions were used to transform two auxotrophic (ura–) S. cerevisiae strains: the protease-deficient mutant BJ5464 (MAT{alpha} ura3 trp1 leu2-112 his3 pep4::his4 prb1 GAL&double_tag;+) and strain DBY746 (MAT{alpha} his3-1 leu2-3 leu2-112 ura3-52 trp1-289 amber). Yeast transformation was carried out by following the method described by Ito et al. (21), in which a previous transformation in E. coli helps to analyze the orientation of clt genes with respect to the GAL1 promoter. After growth on synthetic dextrose medium plates lacking uracil (37), recombinant yeast colonies were analyzed by PCR to confirm the presence of the corresponding gene. Fresh cells were resuspended in Lyse-N-Go PCR reagent (Pierce) and subjected to the following conditions in order to ensure cellular breakage: 65°C for 30 s, 8°C for 180 s, 65°C for 90 s, 97°C for 180 s, 8°C for 60 s, 65°C for 180 s, 97°C for 60 s, and 65°C for 60 s. Finally, 1 µl of lysate was used as the template DNA for an amplification program with the oligonucleotides shown in Table 2.

The recombinant yeast strains isolated were then grown at 30°C and with shaking at 200 rpm in synthetic dextrose medium, supplemented with all amino acids and bases except uracil and containing glucose (2%) as the sole carbon source to ensure growth. When cells had reached the stationary phase of growth, they were washed twice and resuspended in the same medium but with galactose (2%) instead of glucose in order to induce the expression of heterologous genes cloned under the control of the GAL1 promoter.

Samples were withdrawn every 8 h for 48 h, and milk-clotting activity, which first appeared after 24 h of growth, was investigated. Such activity was found only in the intracellular extracts. Yeast cell disruption was performed with a Braun (Melsungen, Germany) MSK CO2-cooled cell homogenizer in the presence of 0.45-mm-diameter glass beads.

Strain BJ5464 (protease deficient) was completely unable to support the expression of the M. xanthus ORFs, while milk-clotting activity was detected when those ORFs (both cltA and cltB together and cltC alone) were expressed in the DBY746 strain, although only at the intracellular level, with cltA and cltB affording firmer milk clots than cltC. Similar results were obtained by Egel-Mitani et al. (10) and Anahit et al. (2), suggesting that clt genes expressed in protease-positive backgrounds may indeed be involved in the induction of other proteases present in the host strains. On the other hand, although the cltC product sequence does not show any degree of similarity to aspartic proteases or to any type of proteases (note that certain bacterial proteases have been included in the aspartic protease group even though they are not inhibited by typical aspartic protease inhibitors and they use glutamic acid instead of aspartic acid as the active residue), the cltC gene product showed the milk-clotting phenotype. This again corroborates the hypothesis that these genes might be inducing the action of other molecules present in the host strain that are somehow involved in proteolysis.

Finally, the genes affording the best results (firmer clots in S. cerevisiae) in milk-clotting activity (cltA and cltB genes) were prepared again by using the above-described constructions and ligated into the pGAPZ{alpha}A vector (Invitrogen) previously digested with EcoRI. The promoter on pGAPZ{alpha}A was fused to the S. cerevisiae {alpha}-factor secretion signal sequence.

After the correct orientation of cltA and cltB with respect to the GAP promoter was checked, plasmids containing inserts in the appropriate orientation were linearized with AvrII and used to transform a P. pastoris strain (his4) as described in Pichia cloning kit (Invitrogen) instructions. The colonies able to grow in yeast extract-peptone-dextrose supplemented with zeocyne (100 µg/ml) were PCR analyzed as described before for S. cerevisiae, by using the oligonucleotides shown in Table 2. Recombinant P. pastoris colonies were then grown on yeast extract-peptone-dextrose with zeocine at 30°C and with shaking at 300 rpm, and milk-clotting activity was subsequently analyzed in both intracellular extracts and in the supernatant broths over 96 h of culture. After 50 h, the recombinant colonies showed intracellular milk-clotting activity, and this ability remained steady until the end of the experiments.

In any case, the secretion signal contained in the pGAPZ{alpha}A vector used for cloning was not sufficient to direct active secretion in P. pastoris.


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Nucleotide sequence accession numbers.
 
The sequences for ORFs determined in this study have been submitted to GenBank under accession numbers AY033401, AY033402, AY033403, AY033404, AY033405, AY033406, AY033407, and AY426316.


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ACKNOWLEDGMENTS
 
We express our gratitude to the Spanish Ministry of Science and Technology and to FEDER for making this work possible.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Parasitology, Faculty of Pharmacy, Campus Sur 15782, Santiago de Compostela, Spain. Phone and fax: 34 981 592490. E-mail: mpvilla{at}usc.es. Back


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Applied and Environmental Microbiology, October 2004, p. 6337-6341, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6337-6341.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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