Fibrinolytic enzyme genes ( aprE2 , aprE176 , and aprE179 ) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy-, Spec ^S ) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang ), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner. Bacillus subtilis and closely related species play important roles for the fermentation of various Asian soy foods, including Korean doenjang , cheonggukjang , and ganjang , Japanese natto , Chinese douchi , and Indonesian gembus [14] . Bacilli secrete amylases and proteases, which are responsible for the degradation of nutrients in soybeans, production of peptides and amino acids, and flavoring compounds [13 , 16] . Some secreted proteases possess fibrinolytic activities and degrade fibrin directly. Nattokinase secreted by some B. subtilis strains is the most well-known example [18] . In recent years, fibrinolytic enzymes from bacilli have been the subject of extensive studies because of their ability to degrade fibrin, the major cause of thrombolytic diseases such as acute myocardial infarction and cerebral infarction [12] . In addition to serving as a source for therapeutic agents, bacilli strains with fibrinolytic activities are useful as starters for fermented soy foods. Cheonggukjang , a Korean fermented soy food, is prepared by inoculation of cooked soybeans with bacilli and following fermentation for 2 days at 37-42℃. Cheonggukjang is a rich source for bacilli and bioactive metabolites, including fibrinolytic enzymes. Cheonggukjang is consumed after being boiled with added condiments. If cheonggukjang is consumed without heat treatment after fermentation, the fibrinolytic activity of cheonggukjang could be enjoyed in addition to the probiotic effect of bacilli. If the fibrinolytic activities of fermented soybean foods are to be increased, Bacillus strains with strong fibrinolytic activities should be used as starters. An efficient method to increase the fibrinolytic activity of a strain is the introduction of a fibrinolytic gene into the strain. However, the introduction of a gene should be conducted by a food-grade way. In most genetic engineering studies, a target gene is introduced together with an antibiotic resistance gene, which is used as a selection marker [4] . However, an antibiotic resistance gene is not allowed if the host organism is used for food fermentation. In previous studies, we characterized strong fibrinolytic enzymes secreted by B. subtilis strains; AprE2 from B. subtilis CH3-5 [7 , 8] and AprE176 from B. subtilis HK176 [9] . We also improved AprE176 by error-prone PCR [9] . In this work, we introduced aprE2 , aprE176 , and aprE179 (a mutant from aprE176 ) into the chromosome of B. subtilis 176 without an antibiotic resistance gene. We measured the fibrinolytic activities of the recombinant strains and prepared cheonggukjang using the integrants. Introduction of a fibrinolytic gene into a nonessential gene on the chromosome of a Bacillus strain via a food-grade manner seems an effective method to improve the fibrinolytic capacity of a Bacillus strain. Bacterial strains and plasmids used in this study are listed in Table 1 . All B. subtilis recombinants were derived from B. subtilis 168. B. subtilis and E. coli DH5α were g rown in LB broth (Luria Bertani broth; Acumedia, Lansing, MI, USA) at 37℃ with aeration. B. subtilis PD3-5nc, PD176nc, and PD179nc were grown in LB containing spectinomycin (100 μg/ml; Sigma, St. Louis, MO, USA). For E. coli cells harboring pDG1662, pDG3-5nc, pDG176nc, or pDG179nc, ampicillin (100 μg/ml, Sigma) was included in the LB medium. AprE2 was amplified from B. subtilis CH3-5 by using a primer pair, aprEFB and aprERS ( Table 2 ). aprE176 was amplified from B. subtilis HK176 by using a primer pair, 51F and 51R-S ( Table 2 ). The PCR conditions were as follows: 94℃ for 5 min, followed by 30 cycles consisting of 94℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 1 min. The aprE179 gene was amplified by splicing overlap extension PCR (SOE-PCR) as described previously [11] . The SOE-PCR was completed through two rounds of PCR. The 179up (1-1,206 bp) and 179down (1,185-1,528 bp) fragments of aprE179 were amplified using the 51F and 179siteR, and 179siteF and 51R-S primer pairs, respectively. One microliter of the first-stage PCR product was used as the template for the second-stage PCR, and the primers 51F and 51R-S were used to amplify the full-length aprE179 . The PCR conditions were as follows: 94℃ for 5 min, followed by the first 10 cycles consisting of 94℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 1 min and the next 20 cycles consisting of 94℃ for 30 sec, 63℃ for 30 sec, and 72℃ for 1 min. The amplified aprE2 , aprE176 , and aprE179 genes were inserted into plasmid pDG1662 after being digested with Bam HI and Sal I ( Fig. 1 ). E. coli DH5α competent cells were prepared and transformed by electroporation as described previously [9] . B. subtilis 168 competent cells were prepared and transformed by the two-step transformation method of Cutting and Vander Horn [3] . The two-step replacement recombination procedures are described in Fig. 2 [17] . In the first step, B. subtilis 168 cells harboring each integration plasmid were cultivated in LB broth containing spectinomycin (100 μg/ml) at 37℃ and integrants were screened on LB plates with spectinomycin. In the second step, an integrant obtained from the first step was cultivated in LB broth without an antibiotic for 18 h at 37℃. Then the temperature was increased to 45℃ and the plates were incubated for the next 24 h. Then spectinomycin-sensitive (Spc ^S ) colonies were screened on LB plates. Colony PCR was used to confirm the structure of Spc ^r clones from the first integration s tage and Spc ^S clones from the second crossover events. A small portion of cells was scraped from a colony and introduced into a 0.2 ml Eppendorf tube containing 10 μl of 2 × PCR mixture (GoTag Long PCR M aster; Promega, Madison, WI, USA). PCRs were done using various primer pairs and the amplification program consisted of 93℃ for 3 min, 35 cycles of 93℃ for 15 sec, 62℃ for 30 sec, and 68℃ for 4 min. After the PCR, 5 μl of each amplified product was analyzed by agarose gel (1% (w/v)) electrophoresis. Cheonggukjang was prepared from soybeans (2013 crop year, Hamyang, Gyeongnam, Korea). Soybeans (300 g) were washed and soaked in water for 15 h at room temperature. After the water was decanted, the whole soybeans were autoclaved for 45 min at 121℃. Soybeans were inoculated with B. subtilis strains (2% inoculum size, dry soybean weight (v/w)): 168 (control cheonggukjang ), SJ3-5nc, SJ176nc, and SJ179nc. Fermentation was proceeded for 3 days at 37℃ and cheonggukjang samples were taken at time points (6, 12, 24, 36, 48, 60, and 72 h) for measuring the cell numbers and fibrinolytic activities. The fibrinolytic activities of culture supernatant and cheonggukjang were assayed by using the fibrin plate method as described previously [7 , 10] . Plasmin (Sigma) was spotted on a fibrin plate at different concentrations (3-40 mU) and the plate was incubated for 18 h at 37℃. The size of the lysis zone was measured using a Vernier caliper and a standard curve was obtained. The protein concentration of a sample was determined by the Bradford method [2] using bovine serum albumin as the standard. All measurements were done in triplicates and the means were represented with standard deviations. 1.7 kb fragment containing gene aprE2 was amplified from the B. subtilis CH3-5 genome. The fragment included the putative promoter sequences and the possible transcription terminator. By the same way, a 1.5 kb aprE176 gene was amplified from B. subtilis HK176. aprE179 , a mutant derived from aprE176 , was obtained by the splicing overlap extension PCR technique. aprE179 differs from aprE176 in a single nucleotide. The 526 ^th nucleotide from the start codon, GTG, is G in aprE176 but A in aprE179 , causing the amino acid change Ala to Thr [9] . Each amplified fragment was cloned into pDG1662 at the Bam HI and Sal I sites, resulting in pDG3-5nc, pDG176nc, and pDG179nc, respectively ( Fig. 1 ). The three integration plasmids had the same structure and a fibrinolytic gene was located in the middle of amyE . B. subtilis 168 was selected as the host for the integration of the fibrinolytic genes because this strain has a basal level of fibrinolytic activity and is easily transformed. Transformation of B. subtilis CH3-5 and B. subtilis HK176, wild-type strains isolated from cheonggukjang , was not successful. Selection of the integrants, where the whole plasmid was integrated into the amyE site of the B. subtilis 168 chromosome, was performed using LB plates containing spectinomycin. Colonies on LB plates with spectinomycin were examined for the plasmid integration by colony PCR ( Fig. 3 ). Amplification of the amyE and spectinomycin resistance genes was carried out. When primers for amyE were used, two bands were amplified from the integrants but a single band was amplified from B. subtilis 168 ( Fig. 3 A, lanes a1-a4). There were two amyE genes in the integrants; one copy was an intact amyE and the other contained aprE in the middle of the gene ( Fig. 2 ). When primers for the spectinomycin resistance gene were used, a 600 bp fragment was amplified from B. subtilis PD3-5nc, PD176nc, and PD179nc but not from B. subtilis 168 ( Fig. 3 A, lanes b1-b4). These results indicated that pDG1662 plasmids containing different fibrinolytic genes were individually integrated into the chromosome of B. subtilis 168 by single crossover. B. subtilis PD3-5nc, PD176nc, and PD179nc, integrants obtained from the first round of single crossover, were forced to undergo the second round of crossover as mentioned in the Methods section. Colonies on LB plates were examined. Recombinants were expected, which were generated by the second homologous recombination between two amyE sequences [6] . Depending upon the location where homologous recombination occurred, two different recombinants were expected ( Fig. 3 ). One was reverted to wild type, B. subtilis 168, and the other was the strain where a fibrinolytic gene remained in the middle of the amyE site but the Spc ^r and Amp ^r genes were deleted ( Fig. 2 ). Colonies on LB agar plates were spotted onto LB plates with 1% soluble starch or spectinomycin (100 μg/ml). After several trials, colonies showing Spe ^S and Amy- were obtained. When PCR was done using the amylF/amylR primer pair, a 1.5-1.7 kb fragment was amplified, whereas a smaller band was amplified from B. subtilis 168 ( Fig. 3 B, lanes a1, a5, and a7). When the amylF/51R-S primer pair was used, 1.6-1.7 kb bands were amplified from the three recombinants but no band was amplified from B. subtilis 168 ( Fig. 3 B, lanes c1, c5, c6, and c7). These colony PCR results together with the observed phenotypes confirmed that the strains were obtained through the 2 ^nd homologous recombination. They were named B. subtilis SJ3-5nc, SJ176nc, and SJ179nc, respectively. Incubation temperature is an important factor to increase the frequency of the second crossover event. The desired recombinants were obtained only after cells were first incubated for 24 h at 37℃ and another 18 h at 45℃. Several researchers used plasmids with temperature-sensitive replication for the integration into the host chromosome because the transformation efficiency is much higher than non-replicating DNA. Inside the host, the plasmid replicates at the permissive temperature but cannot replicate when the temperature increases. Colonies grown on plates with an antibiotic are those cells where the entire plasmid was integrated into the host chromosome. pMAD, a replication-thermosensitive mutant of pE194, was used for two-step gene replacement in some gram-positive bacteria [1] . pNZT1, a plasmid with thermosensitive replication, was used for the replacement of the native glcU - gdh operon promoter with the pur operon promoter in Bacillus amyloliquefaciens [19] . Unlike these vectors, pDG1662 does not replicate in Bacillus species, and it is in fact an E. coli plasmid [5] . Thus, the efficiency for the integration of pDG1662 into B. subtilis 168 chromosome was not high. Still, colonies were obtained that grew on LB with spectinomycin. Integrants where the whole plasmids were inserted into the amyE site of B. subtilis 168 were incubated at different temperatures (37℃, 40℃, and 45℃) and times for the second crossover event. We found a condition under which the frequency of the second single crossover event was increased. By incubating the integrants for 18 h at 37℃ and another 24 h at 45℃, the desired strains were obtained. B. subtilis SJ3-5nc, SJ176nc, and SJ179nc were cultured in LB broth for 144 h and their growth and fibrinolytic activities were measured ( Fig. 4 ). No differences were observed in growth between B. subtilis 168 and its derivatives. The OD ₆₀₀ values reached 1.5-1.6 in 96 h, and then decreased rapidly for all four cultures ( Fig. 4 ). The fibrinolytic activities of the recombinants increased gradually and reached the maximum values at 120 h. The highest fibrinolytic activities of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc were 5.0 ± 0.3, 23.3 ± 1.3, 15.8 ± 1.5, and 17.4 ± 1.0 U/ml, respectively ( Fig. 4 B). B. subtilis SJ3-5nc showed 4.7-fold higher activity than B. subtilis 168. B. subtilis SJ179nc showed 3.5-fold and B. subtilis SJ176nc showed 3.2-fold higher activity than B. subtilis 168. B. subtilis SJ3-5nc, where gene aprE2 was integrated into the chromosome, showed higher activity than those with the aprE176 or aprE179 gene was integrated at the same locus. The fibrinolytic activities of B. subtilis 168 derivatives constructed through this work are less than those of B. subtilis CH3-5 and B. subtilis HK176 (data not shown). One of the reasons is that only a single gene was introduced into B. subtilis 168, but actually many gene products contribute to the fibrinolytic activity of Bacillus strains. B. subtilis CH3-5 and B. subtilis HK176 possess high levels of fibrinolytic activities, but all the responsible enzymes are not well understood, necessitating further research. B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc were individually inoculated into cooked soybeans and cheonggukjang fermentation was performed at 37℃. All four bacilli strains showed good growth, and the viable counts increased rapidly from 10 ⁶ to 10 ⁹ CFU/g of cheonggukjang within the first 6 h ( Fig. 5 ). The pH of cheonggukjang increased from 7.0 to 8.0 after 60 h ( Fig. 5 ). The increase in pH was probably the result of proteolysis and the release of ammonia following the utilization of amino acids by bacilli. Sarkar et al. [15] reported that the increase in pH of soybean fermented with Bacillus sp. DK-WI was coincident with the increase in the proteolytic activity of Bacillus and ammonia concentration during fermentation [15] . The fibrinolytic activities of cheonggukjang remained at basal levels during the first 6 h, and then increased, except for cheonggukjang fermented with B. subtilis 168 ( Fig. 6 ). The fibrinolytic activities increased gradually and reached the highest values at 72 h. The fibrinolytic activity of cheonggukjang fermented with B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 U/g of cheonggukjang , respectively. Cheonggukjang fermented with B. subtilis SJ3-5nc showed the highest fibrinolytic activity. B. subtilis SJ3-5nc also showed the highest activity among the recombinants when grown in LB broth. Cheonggukjang prepared with B. subtilis CH3-5 showed the fibrinolytic activity of 72 U/g at 60 h and cheonggukjang prepared with B. subtilis HK176 showed 55U/g at 48 h (data not shown). The results were not surprising because B. subtilis 168 has a low level of fibrinolytic activity and the introduction of a gene was not enough to increase the fibrinolytic activity drasticially. Because the purpose of this work was to examine the possibility of improving the fibrinolytic activity of Bacillus strains through the introduction of a gene without an antibiotic marker, properties of cheonggukjang such as flavor, texture, and production of metabolites were not examined in detail at this time. Unlike cheonggukjang prepared with B. subtilis CH3-5 or B. subtilis HK176, cheonggukjang prepared with B. subtilis 168 and its integrants did not produce slime materials. As the next step for the production of high-quality cheonggukjang , Bacillus strains conferring good organoleptic properties to cheonggukjang will be selected and their fibrinolytic activities will be improved by the methods shown in this work. The results show a possibility that Bacillus strains can be improved and become more suitable starters for soyfood fermentations. Fibrinolytic enzymes from Bacillus strains are important bioactive compounds that can increase the functionality of fermented foods. Introduction of fibrinolytic genes into Bacillus hosts is an effective way for improving host strains. However, it should be carried out via a food-grade way because an antibiotic resistance gene is not allowed for starters used for food production. In addition to antibiotic resistance markers, food-grade vectors and hosts should not contain any sequences derived from harmful or potentially harmful organisms such as E. coli . In this respect, the strains constructed through this work can be regarded as food-grade hosts because the fibrinolytic genes are derived from B. subtilis strains, which have been used for food fermentations and are considered generally recognized as safe organisms. In the future, construction of strains with higher fibrinolytic activities than strains constructed through this work should be tried. The usefulness of such strains is also checked through soyfood fermentations.