Engineered biosynthesis of cyclic lipopeptide locillomycins in surrogate host Bacillus velezensis FZB42 and derivative strains enhance antibacterial activity
Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity.
KeywordsCyclic lipopeptide Locillomycins Heterologous expression B. velezensis Antibacterial activity
We are grateful to Professors Rainer Borriss (Institut fur Biologie, Humboldt-Universitat zu Berlin, Berlin, Germany) and Xuewen Gao (Nanjing Agricultural University, Nanjing China) for kindly bestowing us with wild-type B. velezensis FZB42 strain. We would like to thank Mr. John Truong and Dr. Xian Zhou (Western Sydney University, Sydney, Australia) for the linguistic revision and critical review of the manuscript.
This study was funded by the National Natural Science Foundation of China (grant 31570061), Special Fund for Agroscientific Research in the Public Interest (201403032), Jiangsu Provincial Department of Education major project (grant 17KJA180001), Jiangsu Agricultural Independent Innovation Fund (grant CX(17)3018), and Six Talents in Jiangsu Province (grant 2016-YY-006).
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Dey G, Bharti R, Ojha PK, Pal I, Rajesh Y, Banerjee I, Banik P, Parida S, Parekh A, Sen R, Mandal M (2017) Therapeutic implication of “Iturin A” for targeting MD-2/TLR4 complex to overcome angiogenesis and invasion. Cell Signal 35:24–36. https://doi.org/10.1016/j.cellsig.2017.03.017. Epub 2017 Mar 24CrossRefGoogle Scholar
- Datsenko KA, Wanner, BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences 97 (12):6640-6645Google Scholar
- Eswari JS, Dhagat S, Kaser S, Tiwari A (2018) Homology modeling and molecular docking studies of bacillomycin and iturin synthetases with novel ligands for the production of therapeutic lipopeptides. Curr Drug Discov Technol 15:132–141. https://doi.org/10.2174/1570163814666170816112536 CrossRefGoogle Scholar
- Fan B, Chen XH, Budiharjo A, Vater J, Borriss R (2011) Efficient colonization of plant roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green fluorescent protein. Biotechnol 151:303–311. https://doi.org/10.1016/j.jbiotec.2010.12.022 Google Scholar
- Götze S, Herbst-Irmer R, Klapper M, Görls H, Schneider KRA, Barnett R, Burks T, Neu U, Stallforth P (2017) Structure, biosynthesis, and biological activity of the cyclic lipopeptide anikasin. ACS Chem Biol 12:2498–2502. https://doi.org/10.1021/acschembio.7b00589. Epub 2017 Sep 1CrossRefGoogle Scholar
- Luo C, Zhou H, Zou J, Wang X, Zhang R, Xiang Y, Chen Z (2014) Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and the biocontrol of rice sheath blight induced by Rhizoctonia solani. Appl Microbiol Biotechnol 99:1897–1910. https://doi.org/10.1007/s00253-014-6195-4 CrossRefGoogle Scholar
- Mountford S, Mohanty B, Roberts KD, Yu HH, Scanlon MJ, Nation RL, Velkov T, Li J, Thompson PE (2017) The first total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity. Org Biomol Chem 15:7173–7180. https://doi.org/10.1039/c7ob01493g CrossRefGoogle Scholar
- Robbel L, Marahiel MA (2010) Daptomycin, a bacterial lipopeptide synthesized by a nonribosomal machinery. J Biol Chem 285:27501–27508. https://doi.org/10.1074/jbc.R110.128181
- Romero D, de Vicente A, Rakotoaly RH, Dufour SE, Veening J-W, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Pérez-García A (2007) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant-Microbe Interact 20:430–440CrossRefGoogle Scholar