Identification of novel surfactin derivatives from NRPS modification of Bacillus subtilis and its antifungal activity againstFusarium moniliforme

Bacillus subtilis strain PB2-L1 produces the lipopeptide surfactin, a highly potent biosurfactant synthesized by a large multimodular nonribosomal peptide synthetase (NRPS). In the present study, the modules SrfA-A-Leu, SrfA-B-Asp, and SrfA-B-Leu from surfactin NRPS in B. subtilis BP2-L1 were successfully knocked-out using a temperature-sensitive plasmid, pKS2-mediated-based, homologous, recombination method. Three novel surfactin products were produced, individually lacking amino acid Leu-3, Asp-5, or Leu-6. These surfactins were detected, isolated, and characterized by HPLC and LC-FTICR-MS/MS. In comparison with native surfactin, [∆Leu3]surfactin and [∆Leu6]surfactin showed evidence of reduced toxicity, while [∆Asp5]surfactin showed stronger inhibition than native surfactin against B. pumilus and Micrococcus luteus. These results showed that the minimum inhibitory concentration of [∆Leu6]surfactin for Fusarium moniliforme was 50 μg/mL, such that [∆Leu6]surfactin could lead to mycelium projection, cell damage, and leakage of nucleic acids and protein. These factors all contributed to stimulating apoptosis in F. moniliforme. The present results revealed that [∆Leu6]surfactin showed a significant antifungal activity against F. moniliforme and might successfully be employed to control fungal food contamination and improve food safety.


Background
Fusarium moniliforme mainly contaminates maize, sorghum, wheat, cotton, beans, tomatoes, peanuts, bananas, beans, peppers, and some feeds. Among these materials, maize is the most prone to fungal infection, accounting for almost 90 % of all types of food pollution [1,2]. As one of the most common fungi, Fusarium mycotoxin researchers are currently most concerned about F. moniliforme. Currently, surfactins are used for their antibacterial, antiviral, anti-tumor, and hemolytic activities [3][4][5][6]. However, surfactins do not only inhibit filamentous fungi, but C15 surfactin has a synergistic inhibition effect on filamentous fungi. The lipopeptide surfactin family has a ring structure peptide chain and possesses a β-hydroxy fatty acid chain (typically C 13 -C 16 ) containing seven amino acids formed by crosslinking [7]. There has been great interest in these compounds because of their potential biological activities as well as economic value. Lipopeptides are often composed of seven or fewer modules composed of amino acids components. Surfactin consists of a Glu-Leu-Leu-Val-Asp-Leu-Leu peptide, synthesized by large multifunctional nonribosomal peptide synthetases (NRPSs) via the multiple thiotemplate mechanism [8,9]. The composite module can be modified by epimerization, methylation, acylation, or cyclization. The final lipopeptide products can have linear, cyclic, or branched peptide backbones [10].
In this study, a procedure is described that allows for efficient and relatively fast inactivation of a Bacillus subtilis gene to create new, biotechnologically interesting products. The approach is the same as developed has been for some other Gram-positive strains [11,12] and uses a high temperature-sensitive, shuttle plasmid based on the pKS2 replication origin. Plasmid pKS2 replicates at 30°C, but 37°C is nonpermissive for plasmid replication. This method is different from the traditional twostep knockout method [13] and can quickly knock out a module with precision. In contrast, the two-step method usually cannot avoid the impact of an exogenous antibiotic resistance gene.
B. subtilis strain BP2-L1 produces surfactin following the integration of genes sfp and degQ into the B. subtilis BP2 chromosome [14]. For knock out of the modules SrfA-A-Leu, SrfA-B-Asp, and SrfA-B-Leu of surfactin NRPSs in B. subtilis BP2-L1, the pKS2-mediated, temperaturesensitive, homologous recombination method was used. The structures of the resulting novel surfactins were identified and isolated to develop new antibacterial lipopeptides with stronger antimicrobial activity and more beneficial characteristics.

Strains, plasmids, and media
Strains and plasmids used in this study are listed in Table 1. B. subtilis strain PB2-L1, a derivative of B. subtilis 168 (trpC2) developed to produce surfactin [14], was used as the source of surfactin synthetase genes and for engineering surfactin synthetase. B. subtilis PB2 was a model strain of Bacillus subtilis from Chester Price' lab of UCDavis. pMD19T-simple vector was a commercial carrier and pKS2 vector was temperature sensitive vector for gene deletion. Escherichia coli DH5α was used for cloning procedures and propagation of plasmids; pKS2-based vectors can be replicated at 30°C in E. coli. Before transforming B. subtilis, plasmids were purified from E. coli strain JM110 to obtain the unmethylated forms. Bacterial cells were cultivated in Luria broth (LB, 5 g yeast extract/L, 10 g peptone/L, and 10 g NaCl/L) or in Landy medium [15] supplemented with 0.1 % yeast extract and 2 mg/L phenylalanine [16], at temperatures of 28 or 37°C. Ampicillin was added to 100 μg/mL.

B. subtilis strain construction
Traditional chemical transformation was used in B. subtilis strain construction. The genotypes of new transformants were identified via PCR. B. subtilis PB2-L1 transformed with the temperature-sensitive vectors pKS2-srfA-C-ΔLeu, pKS2-srfA-B-ΔAsp, and pKS2-srfA-B-ΔLeu. The host strain E. coli JM110 can modify the shuttle vector pKS2 by demethylation and, by modifying demethylation, the rate of B. subtilis transformation can be highly improved. New transformants possess erythromycin resistance, such that these transformants were selected on LB medium agar plates with 10 μg/mL erythromycin [18].
Surfactin is a lipopeptide of seven modules that are assembled by NRPS A-, PCP-, C-, and modifying domains (Fig. 1). This antibacterial lipopeptide must be linearly arranged, synthesized, and cyclized into the final assembly of seven amino acids and a β-hydroxy fatty acid chain. Knocking out one of the modules in NRPS gene clusters produces a lipopeptide lacking one amino acid. Gene exchange is a two-step replacement procedure, as described previously [19]. Resistance genes were replaced in two steps and recombinant strains sensitive to antibiotics (Fig. 2). In the first step, the gene recombinant plasmid from B. subtilis strains was cultured in LB medium at 37°C. The homologous sequences were within the target gene plasmid and the entire plasmid inserted into the genome via a single crossover. In Fig. 2, crossed lines indicate the position of a single crossover and the diagonal block and little dots indicate homologous fragment positions. In the second step, the above-described integrands took place in a single exchange; the second exchange occurred on the chromosome in a parental or homologous sequence at 30°C within 48 h and finally expelled the plasmid [20]. In Fig. 2, the diagonal block on the plasmid and genome was successfully integrated by the initial and second single crossovers, such that the little dots in the block occurred between the plasmid and genome. Colonies with deleted surfactin modules exhibited the erythromycin sensitive phenotype, which was then used to detect the desired genotype. Finally, sensitive clones were obtained and verified by PCR. These erythromycin gene of knockout mutants could not amplify this resistance gene but could amplify upstream and downstream sequences of homologous gene knockout mutants (amplified sequence could not contain the knockout gene sequence).

Culture conditions for obtaining surfactins
B. subtilis strains were inoculated into 250-mL flasks containing 100 mL of LB medium and cultured at 37°C for 24 h with 180 rpm shaking as a preculture. A 5 % (by vol) preculture was inoculated into a 500-mL flask containing 200 mL of Landy medium [15], and the wildtype and modified surfactin produced by culturing at 30°C with 180 rpm shaking for 72 h.

Extraction of a novel surfactin
After cultivation, a culture was centrifuged at 11000 × g for 15 min to remove bacterial cells. The supernatant pH was adjusted to 2.0 by adding 6 N HCL until the supernatant produced a precipitate. The supernatant was then centrifuged at 11000 × g for 10 min and the precipitate collected [21]. The precipitate was then resuspended in methanol several times with subsequent centrifugation. Finally, the pH was adjusted to 7.0 using 6 N NaOH to obtain an extract dissolved in methanol.

Identification of a novel surfactin
A lipopeptide surfactin was extracted as described previously [22]. The extracts were analyzed by HPLC/MS (Hewlett Packard 1100 Series C8 column, Hewlett-Packard Co., Palo Alto, CA, USA) and monitored at 210 nm as well as in negative-ion mode over the m/z range from 500 to 1200. The solvent gradient profile used buffers A and B (0.05 % aqueous formic acid and 0.045 % methanolic formic acid, by vol, respectively) at a flow rate of 0.3 mL/min, with sample elution starting with 70 % buffer B, followed by a linear gradient to 100 % buffer B over 30 min. All samples were analyzed by continuous infusion into the LTQ® 7 Tesla FTICR mass spectrometry ion trap (LTQ-FT, Thermo Fischer Scientific Inc., Waltham, MA, USA) equipped with a Triversa Nanomate nanoESI ion source (Advion Biosciences Corp., Ithaca, NY, USA) [23]. Samples were detected under negative ion mode (mass range, 150-1500) using the following parameters: ion spray voltage at 2.5 kV, sheath gas at 20 units, capillary temperature at 300°C, capillary voltage at 41 V, and tube lens at 110 V. Xcalibur software was used for visualization of high-resolution spectral profile data (Thermo Fischer Scientific, Inc., 2nd Edition SP2) and the exact patterns of m/z values obtained.

Biological activity analysis of novel surfactin
Surfactin hemolytic activity was analyzed using blood-agar plates [24]. In brief, activity was detected on commercial blood agar using the Oxford Cup for hemolytic activity detection. The ability of products from bacterial strains to inhibit the growth of various indicator organisms by the agar well diffusion method was a qualitative determination [25]. Pre-poured agar media plates were spread with 10 7 CFU/ mL of the respective indicator organism and allowed to dry. Wells of 6.8 mm diameter were cut in the plates using a sterile steel borer and filled with 24 h LB culture filtrate (60 μL) of each isolate. After incubation under appropriate conditions, the diameter of the inhibition zone was measured by using calipers. B. pumilus strains were mixed in LB agar plates, and surfactin A and one of three novel surfactin extracts added into the plates using the 6.8-mm punch method to detect lipopeptide antibiotic activity. Filamentous growth of F. moniliforme were mixed in potato dextrose agar (PDA) plates, and antibacterial activity measured as described previously. Biologically active substances were isolated and purified by HPLC to yield relatively pure products. Under vacuum conditions, the same concentration ratio and sample concentration were obtained for these isolates and the biological activity analyzed. The diameters of cleared zones were measured and are shown in Table 3. Each sample was treated in triplicate and the results expressed as means ± SD.
According to the National Committee for Clinical Laboratory Standards (NCCLS), the minimum inhibitory concentrations (MICs) of purified novel surfactin were detected by the 96-plate microbroth method [26]. Final concentrations of novel surfactin in mixtures ranged from 12.5 to 800 μg/mL. MICs were measured using an ELISA analyzer after incubation of bacteria at 37°C for 20 h and the fungus at 28°C for 24 h.   6 ]surfactin were separately added into PDA medium. Then, 5-mm hyphae discs of F. moniliforme were placed at the PDA media centers. The diameters of the hyphal extent were detected by the decussation method after incubation for 7 d at 28°C and then the inhibition ability rate calculated. At the same time, after washing hyphae of F. moniliforme using 10 mL of saline solution containing Tween 20 (0.1 % by vol), the spore numbers were detected using a hemocytometer. A sample with sterile PBS was used as a control and each group was evaluated in triplicate.

Influence of F. moniliforme hyphae by [ΔLeu 6 ]surfactin
Cells at 1 × 10 6 cell/mL of F. moniliforme were cultured on individual PDA culture plates at 28°C for 5 d. Mycelia were harvested from cultures and prepared by washing with sterile saline solution (0.85 % NaCl, by wt) and then centrifuged at 3,000 × g for 3 min. Then, hyphae were picked from PDA plates, placed on the concave side, and [ΔLeu 6 ]surfactin solution added to a final concentration of 50 μg/mL. The hyphae were cultured in moisturizing gauze on Petri dishes and, after incubation for 1, 2, or 4 h at 28°C, samples were collected for inspection by ordinary optical microscopy. Sterile PBS was used as a control.

SEM and TEM
Hyphae were washed in 0.1 M phosphate buffered saline (PBS, pH 7.2), and fixed using 2.5 % glutaraldehyde at 4°C for 24 h. The resulting sediments were rinsed three times with 0.02 M PBS, fixed with 2 % osmium tetraoxide for 2 h at room temperature, and dehydrated through a sequence of 30, 50, 70, and 90 % aqueous ethanol solutions. Morphological observations of samples were carried out using a scanning electron microscope (Hitachi High Technologies America, Inc., Shaumburg, IL, USA Inc.) operating at 30.0 kV.

Spore analysis by flow cytometry
Spore suspensions of F. moniliforme at 1 × 10 6 cell/mL were provided with [ΔLeu 6 ]surfactin to a final concentration of 12.5, 25, or 50 μg/m and cultured at 28°C for 1-2 h. After incubation, the spores were collected, centrifuged (5000 × g for 3 min), and washed twice with 0.02 M PBS. The retrieved spores were suspended, adjusted to 1 × 10 6 cell/mL, dyed by adding 10 μL of 1 mg/ mL of propidium iodide solution, and then incubated at 4°C in darkness for 15 min [28]. After incubation, the supernatant was retrieved from samples by centrifugation at 5000 × g for 3 min. Finally, the samples' OD 260 and OD 280 levels were measured to assess the leakage of nucleic acids and proteins from F. moniliforme [29].

Statistical analysis
Statistical analyses were determined using SPSS software (SPSS version 17.0, IBM Corp., Armonk, NY, USA). All experiments were performed in triplicate and data expressed as mean ± standard deviation (SD). A p value of <0.05 was considered significant.

Reconstitution of novel surfactin synthetase
The first step in SrfA-A and SrfA-B subunit rearrangement was the deletion of D-Leu-, Asp-, and D-Leumodules. Deletion of the SrfA-A-Leu, SrfA-B-Asp, or SrfA-B-Leu modules in the srfA-A or srfA-B subunit was completed using the marker-free method (Fig. 2). In the first deletion, a 4.4 kb fragment of SrfA-A-Leu was deleted with an upstream and downstream integration by means of the temperature sensitive shuttle plasmid pKS2-SrfA-A-ΔLeu. The deleted fragment located between 1050 and 2090 (the deleted fragment of srfA-A subunit corresponding to amino acid positions) in the srfA-A subunit. The second deletion mutant was produced using the disruption plasmid pKS2-SrfA-B-ΔAsp. This disruption plasmid deleted the SrfA-B-Asp module from position 1196 to 2092, which encoded an L-Asp-incorporating module in the srfA-B subunit. The third module mutant introduced a deletion in the SrfA-B-Leu module from position 2093 to 3574, which encoded the D-Leu-incorporating module in the srfA-B subunit. The upstream and downstream sequences of the deleted fragment were amplified using PCR, using the corresponding primers, and the occurrence of homologous recombination demonstrated. The resulting plasmid-less clones were screened for the desired modifications using colony PCR analysis. In deletion mutants, the erythromycin resistance gene in the chromosome was removed at the last step and, thus, the erythromycin resistance gene could not be amplified from the deletion mutants by PCR.
Using amplification primer pairs, 5′srfA-A-ΔLeu-up-F and 3′srfA-A-ΔLeu-down-R, and 5′pKS-1058-ERM-F and 3′pKS-1058-ERM-R (Table 2), the target band was 1107 bp, which described mutants containing the upstream and downstream sequence of the Leu module. PCR validation confirmed that, at the molecular level, the deleted module sequence was indeed not in the mutant genome. The deletion of SrfA-B-Asp and SrfA-B-Leu modules was also in accordance with the above methods. The lipopeptide biosyntheses of mutant B. subtilis LS1, LS6 and LS9 were compared and HPLC results showed that the novel surfactin yields from these mutant strains were relatively low. Thus, the three novel surfactin structures were analyzed and identified by FTICR-MS, a highly sensitive detection method.
The molecular mass of surfactin A in the range m/z 1008-1036 was similar to previous published molecular masses (Fig. 3a) [31], and its fatty chain length was 13-15 carbon atoms. The molecular mass of [ΔLeu 3 ]surfactin was in the range of m/z 909-937 (Fig. 3b). The other two novel surfactins, [ΔAsp 5 ]surfactin and [ΔLeu 6 ]surfactin, were also confirmed by mass spectra of ions at m/z 907-935 and 909-937 (corresponding to H + adducts), respectively ( Fig. 3c  and d). Overall the chain lengths of these three novel surfactin derivatives were in the range of 14 to 16 carbon atoms, such that surfactin A and the three novel surfactin derivatives showed different chain lengths in their β-hydroxy fatty acids.  Fig. 3c and d). FTICR-MS/MS results indicated that fracture fragments of these three novel surfactin were consistent with the predicted patterns.

Bioactivity analysis of novel surfactins
Lipopeptide surfactin A causes hemolysis and inhibits a broad range of microorganisms, but it does not inhibit filamentous fungi. Blood agar plates and surfactin extracts from B. subtilis PB2-L1, LS1, LS6, and LS9 were cocultured at 37°C for 24 h. Hemolytic activity was detected using the Oxford Cup method. [ΔLeu 3 ]surfactin produced a clear hemolytic zone surrounding extracts of culture supernatants (Fig. 4a). In contrast, no hemolysis was visible for [ΔLeu 3 ]surfactin and [ΔLeu 6 ]surfactin. However, the hemolytic zone of methanol extracts from B. subtilis LS6 was bigger than that for B. subtilis PB2-L1.
Growth inhibitions of microorganisms by the three novel surfactins were compared by incubating the mixtures at 37°C for 1 d. The inhibition zone of [ΔAsp 5 ]surfactin was very obvious, presenting a clear, transparent   (Table 3). For example, the colony diameters of Fusarium moniliforme were 13.12 ± 0.49 mm for [ΔLeu 6 ]surfactin, and the surfactin showed no inhibition of F. moniliforme hyphae growth.
As it can be seen from Table 4, a molecular recombinant method was employed to alter the surfactin gene cluster, and the properties of the resulting novel surfactins were found to be altered, after production by fermentation. [ΔLeu 6 ]surfactin exhibited the ability to inhibit fungi, and the MIC of [ΔAsp 5 ]surfactin was higher than surfactin A. The MIC of [ΔLeu 6 ]surfactin for F. moniliforme was 50 μg/mL. These results revealed that the lack of leucine in surfactin reduced surfactin hemolytic activity while retaining antibacterial activity. At the same time, hemolytic and antibacterial activities were increased because of the lack of aspartic acid in surfactin.

Inhibitory ability of [ΔLeu 6 ]surfactin for F. moniliforme hyphae and spores
The results shown in Table 5

Influence of F. moniliforme hyphae by [ΔLeu 6 ]surfactin
Compared with control hyphae, many small vesicles were observed on numerous mycelia after treatment with 50 μg/mL [ΔLeu 6 ]surfactin for 1 h (Fig. 5b); there were no observable vesicles in control hyphae. After   (Fig. 5d), the abnormal central mycelial enlargements were ruptured and mycelia appeared as thin strips. This phenomenon was not observed in controls (Fig. 6a). F. moniliforme hyphal structures were observed by SEM and TEM (Fig. 6). Hyphae treated without [ΔLeu 6 ]surfactin grew normally with a straight, smooth appearance (Fig. 6a), while bending and rough structures were observed after treatment with 50 μg/mL of [ΔLeu 6 ]surfactin (Fig. 6b). TEM images of growing, healthy, and normal hyphae, treated without [ΔLeu 6 ]surfactin, showed smooth surfaces and all cellular organelles were visible and in normal arrangements (Fig. 6c). In contrast, although hyphal structures remained intact when treated with 50 μg/mL [ΔLeu 6 ]surfactin, organelles were gathered in clumps and some large vacuoles were noticeable in their central regions (Fig. 6d). SEM and TEM observations indicated that [ΔLeu 6 ]surfactin clearly affected F. moniliforme growth.

Impact of [ΔLeu 6 ]surfactin on F. moniliforme spores
In samples treated for 1 h with a final concentration of 12.5 μg/mL [ΔLeu 6 ]surfactin, a portion of F. spores were already stained with fluorescent dye (Fig. 7). With increased surfactin concentration, fluorescent dye staining rapidly increased. Two-hour samples treated with the same concentration did not show increased fluorescent dye spore staining. However, in 25 and 50 μg/mL [ΔLeu 6 ]surfactin treated samples, staining of spores increased significantly. The increase in the number of spores was determined by the intrusion of fluorescent dyes. Thus, these observations revealed that [ΔLeu 6 ]surfactin's effects on F. moniliforme spores also led to damage or apoptosis of spores.

Impact of [ΔLeu 6 ]surfactin on the integrity of F. moniliforme mycelia
The effects of a surfactin on mycelium integrity can be reflected in the release of intracellular contents, such as an increased release of nucleic acid and proteins (assessed by changes in OD 260 and OD 280 , respectively).
After treatment of F. moniliforme with 25 and 50 μg/ mL of [ΔLeu 6 ]surfactin, F. cell nucleic acids and proteins were rapidly released into the external bacterial body, detected by spectroscopic absorption analysis of released nucleic acids and proteins (Fig. 8). The OD 260 and OD 280 of extracellular fluids increased rapidly in samples treated for 2 h with 50 μg/mL [ΔLeu 6 ]surfactin. After

DNA binding assay
With increased antimicrobial lipopeptide concentrations, the binding capacity of F. moniliforme DNA gradually strengthened, indicating that [ΔLeu 6 ]surfactin imposed a certain effect on the fungal genome. As shown in Fig. 9, treatment of fungal samples with 200 μg/mL [ΔLeu 6 ]surfactin resulted in the Fusarium genome DNA showing some dispersion. These results showed that surfactin possessed the ability to bind to fungal DNA, with [ΔLeu 6 ]surfactin exhibiting the greatest such ability.

Discussion
In this study, a method for marker-free knockout of surfactin synthase was adopted and surfactin synthase successfully altered in three different modules. These modules were responsible for the synthesis of leucine and aspartic acid. Some deletion module surfactin derivatives have already been demonstrated, including versions in which the second and last modules of surfactin synthetase were deleted [32,33]. Schneider et al. have previously reported a different modification method, which relied on molecular surgery within the domains to achieve an exchange of A-units [22]. This approach has been successfully applied to position 7 in surfactin NRPS [34]. From previous reports, the module containing the    6 ]surfactin, and then the reaction mixtures after incubating for 1 h at room temperature were performed to 1 % agarose gel electrophoresis epimerization domain has never before been removed. The comS regulation factor is the key factor in forming competent B. subtilis cells. As the comS regulatory factor is contained in the aspartate module, it is difficult to delete this module. Here, it is reported for the first time the complete deletion of this module containing this regulatory factor.
The purification method for novel surfactins draws on experience with a former method for surfactin extraction and purification [35]. For detection of biological activity, the hemolytic activity of [ΔAsp 5 ]surfactin was found to have strengthened antimicrobial activity. For filamentous fungi, no inhibition zones have been discovered. These results revealed that this method for structural transformation of surfactin was feasible and practical.
However, biological activity tests for novel surfactins lacking D-leucine showed that the hemolytic activities of [ΔLeu 3 ]surfactin and [ΔLeu 6 ]surfactin were significantly decreased compared with the original surfactin A.
[ΔLeu 6 ]surfactin drastically inhibited the growth of F. moniliforme hyphae and spores when the [ΔLeu 6 ]surfactin was at 50 μg/mL. Therefore, it was evident that [ΔLeu 6 ]surfactin significantly inhibited F. moniliforme growth. Surfactin is a well-known lipopeptide biosurfactant with antimicrobial activity, but there is little knowledge regarding surfactin's antifungal activity [36]. Surfactins are not alone in inhibiting filamentous fungi, but C15 surfactin and antifungal drugs have been reported to have a synergistic effect. Surfactins have two polar amino acid residues, such as Glu and Asp, and have been concluded to bind with DNA via hydrogen bonds [37,38].
SEM and TEM observations indicated that [ΔLeu 6 ]surfactin obviously affected F. moniliforme growth by causing morphological changes in hyphae, suggesting that [ΔLeu 6 ]surfactin markedly contributed to inhibiting fungal growth. DNA binding results indicated that [ΔLeu 6 ]surfactin negatively influenced the maintenance of DNA integrity by binding to F. moniliforme DNA, which might in turn genetically affect DNA function for F. moniliforme growth.
This provides the possibility for a new surfactin with potential for food industry applications. As the original surfactin A has relatively strong hemolytic activity, its use has been restricted in the food industry. The purpose of this study was to develop a novel class of antibacterial lipopeptides from surfactin that possessed reduced cytotoxicity with no significant reduction in antimicrobial activity.
The inhibitory ability of filamentous fungi by three novel surfactins was examined here. Very interestingly, [ΔLeu 3 ]surfactin and [ΔLeu 6 ]surfactin exhibited the ability to inhibit filamentous fungi. These findings extended the known properties of surfactin derivatives as well as related changes in antifungal activity. Further research is needed into the mechanism of this antifungal inhibition.

Conclusion
In this study, molecular biological tools were successfully employed to transform surfactin synthase and produce three new substances, [ΔLeu 3 ]surfactin, [ΔAsp 5 ]surfactin, and [ΔLeu 6 ]surfactin. Analyses of these substances' biological activity showed that [ΔLeu 3 ]surfactin and [ΔLeu 6 ]surfactin possessed significantly reduced hemolytic activity but with the concurrent appearance of inhibitory ability for filamentous fungi.