Mutations in genes encoding antibiotic substances increase the synthesis of poly-γ-glutamic acid in Bacillus amyloliquefaciens LL3

Abstract

Poly-γ-glutamic acid (γ-PGA) is an important natural biopolymer that is used widely in fields of foods, medicine, cosmetics, and agriculture. Several B. amyloliquefaciens LL3 mutants were constructed to improve γ-PGA synthesis via single or multiple marker-less in-frame deletions of four gene clusters (itu, bae, srf, and fen) encoding antibiotic substances. γ-PGA synthesis by the Δsrf mutant showed a slight increase (4.1 g/L) compared with that of the wild-type strain (3.3 g/L). The ΔituΔsrf mutant showed increased γ-PGA yield from 3.3 to 4.5 g/L, with an increase of 36.4%. The γ-PGA yield of the ΔituΔsrfΔfen and ΔituΔsrfΔfenΔbae mutants did not show a further increase. The four gene clusters' roles in swarming motility and biofilm formation were also studied. The Δsrf and Δbae mutant strains were both significantly defective in swarming, indicating that bacillaene and surfactin are involved in swarming motility of B. amyloliquefaciens LL3. Furthermore, Δsrf and Δitu mutant strains were obviously defective in biofilm formation; therefore, iturin and surfactin must play important roles in biofilm formation in B. amyloliquefaciens LL3.

Keywords: antibiotic substance; biofilm formation; gene marker-less deletion; poly-γ-glutamic acid; swarming.

Figure 1

(A) Schematic of modular engineering approach in Bacillus amyloliquefaciens LL3 strain. The X marks indicate the gene deletions in the optimized pathway. (B) Condensed structural formulae of the three cyclic lipopeptides, surfactin, iturin A, and fengycin, produced by B. amyloliquefaciens LL3

Figure 2

Confirmation of the deletion of the genes by agarose gel electrophoresis of PCR products with primers BaeOUT‐F/R (lanes 1 and 2), SrfOUT‐F/R (lanes 3 and 4), ItuOUT‐F/R (lanes 5 and 6), and FenOUT‐F/R (lanes 7 and 8). Chromosomal DNA of the mutant strains served as the template for PCR. Fragments of the wild‐type strain were too long to obtain PCR products

Figure 3

Swarming experiments of the wild‐type strain, B. amyloliquefaciens LL3Δsrf, LL3Δfen, LL3Δbae, LL3Δitu, LL‐IS (ΔituΔsrf), LL‐ISF (ΔituΔsrfΔfen), and LL‐ISFB (ΔituΔsrfΔfenΔbae). Strains were observed after 24‐hr cultivation on LB medium with 0.5% agar

Figure 4

Biofilm formation of the wild‐type strain, B. amyloliquefaciens LL3Δsrf, LL3Δitu, LL3Δbae, LL3Δfen, LL‐IS (ΔituΔsrf), LL‐ISF (ΔituΔsrfΔfen), and LL‐ISFB (ΔituΔsrfΔfenΔbae)

Figure 5

(A) Comparison of culture viscosity, DCW, and γ‐PGA yield of the wild‐type strain and the mutant strains carrying single‐gene deletion (B. amyloliquefaciens LL3Δsrf, LL3Δbae, LL3Δfen, and LL3Δitu) after 48 hr of cultivation; (B) Comparison of culture viscosity, DCW, and γ‐PGA yield of the wild‐type and mutant strains carrying multiple deletions (B. amyloliquefaciens LL‐IS, LL‐ISF, and LL‐ISFB) after 48 hr of cultivation

Figure 6

HPLC‐MS spectrograms of standard surfactin and surfactin produced by the wild strain and LL3Δsrf. (A, B) HPLC and MS spectrograms of standard surfactin. (C, D) HPLC and MS spectrograms of surfactin from the wild strain. (E, F) HPLC and MS spectrograms of the LL3Δsrf culture broth, which was disrupted in srf cluster and deficient in production of surfactin