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

Abstract

Background: 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.

Results: 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, [∆Leu(3)]surfactin and [∆Leu(6)]surfactin showed evidence of reduced toxicity, while [∆Asp(5)]surfactin showed stronger inhibition than native surfactin against B. pumilus and Micrococcus luteus. These results showed that the minimum inhibitory concentration of [∆Leu(6)]surfactin for Fusarium moniliforme was 50 μg/mL, such that [∆Leu(6)]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.

Conclusions: The present results revealed that [∆Leu(6)]surfactin showed a significant antifungal activity against F. moniliforme and might successfully be employed to control fungal food contamination and improve food safety.

Fig. 1

the surfactin A biosynthesis gene cluster. The surfactin synthetases A is composed of SrfA-A, SrfA-B, SrfA-C and SrfA-TE, respectively (a). The assembly line of surfactin in the genome consists of three polycistronic genes srfA-ABC (b) which can be further subdivided into five functional domains (c). The dotted boxes indicate the three modules we deleted in this work

Fig. 2

Knockout strategy of marker-free gene. Note that recombination may occur, both at the upstream fragment of the target gene, as shown below, and at the downstream fragment. In both cases, the final connection result of the chromosome is the same thing. All of intermediation process was identified by polymerase chain reaction

Fig. 3

A. FTICR-MS of lipopeptides produced by B. subtilis. (A) B. subtilis PB2-L1, (B) B. subtilis LS1, (C) B. subtilis LS6 and (D) B. subtilis LS9. The values 1008.7, 1022.7 and 1036.7 correspond to the calculated H⁺ adducts of surfactin A (A) with a fatty acid residue ranging from 13 to 15 carbon atoms. The values 909.6, 923.6 and 937.6 correspond to the calculated H⁺ adducts of [∆Leu³]surfactin (B) with fatty acid bodies ranging from 14 to 16 carbon atoms. The values 907.6, 921.7 and 935.7 correspond to the calculated H⁺ adducts of [∆Asp⁵]surfactin (C) with fatty acid bodies ranging from 14 to 16 carbon atoms. The [∆Leu⁶]surfactin values (D) are the same as the [∆Leu³]surfactin values (B). The (b, c, d) shows FTICR-MS/MS of [∆Leu³]surfactin, [∆Asp⁵]surfactin, [∆Leu⁶]surfactin. B. The proposed structure of engineered Surfactin A produced by B. subtilis

Fig. 4

Analysis of bioactivity activity of novel surfactins. a The activity of hemolysis after incubation on blood agar plates for 24 h at 37 °C. b Inhibition of Bacillus pumilus after incubation on LB agar plates for 12 h at 37 °C. (C) Inhibition of Fusarium moniliform after incubation on PDA plates for 48 h at 28 °C. a was the hemolysis test; (b) and (c) were the antibacterial and antifungal test

Fig. 5

800 × micrographs of optical microscope for hyphe of Fusarium moniliforme treated by [∆Leu⁶]surfactin. a the hypha treated without [∆Leu⁶]surfactin; (b) the hypha treated with 50 μg/mL of [∆Leu⁶]surfactin for 1 h; (c) the hypha treated with 50 μg/mL of [∆Leu⁶]surfactin for 2 h; (d) the hypha treated with 50 μg/mL of [∆Leu⁶]surfactin for 4 h

Fig. 6

SEM and TEM micrographs of hyphae of Fusarium moniliforme treated by [△Leu⁶]surfactin. a, the hyphae treated without [△Leu⁶]surfactin (SEM); b, the hyphae treated with 50 μg/mL of [△Leu⁶]surfactin (SEM); c, the hyphae treated without [△Leu⁶]surfactin (TEM); d, the hyphae treated with 50 μg/mL of [△Leu⁶]surfactin (TEM)

Fig. 7

cell damage of Fusarium moniliforme spores by flow cytometry. a. flow cytometry graphs of Fusarium moniliforme spores treated by 0, 12.5, 25, 50 μg/mL of [∆Leu⁶]surfactin. M1 and M2, events of Fusarium moniliforme spores dead cells, 10 000 cells were detected. b. Statistical analysis of [∆Leu⁶]surfactin for cell damage of Fusarium moniliforme spores, Asterisk letters indicated significant differences between control group and treatment group. (*P value < 0.05, **P value < 0.01).

Fig. 8

Divulgation of nucleic acids and proteins of Fusarium moniliforme. CK, samples treated without surfactin. Divulgation of nucleic acids (OD₂₆₀) (a) and proteins (OD₂₈₀) (b) were calculated (*P value < 0.05, **P value < 0.01). The concentration of [∆Leu⁶]surfactin was 25 and 50 μg/mL. The time of treatment reached 3 hours

Fig. 9

DNA binding assay by [∆Leu⁶]surfactin and surfactin. CK, Fusarium moniliforme DNA was mixed with PBS as control. Fusarium moniliforme DNA was mixed with different amounts of surfactin and [∆Leu⁶]surfactin, and then the reaction mixtures after incubating for 1 h at room temperature were performed to 1 % agarose gel electrophoresis