Applied Microbiology and Biotechnology

, Volume 103, Issue 11, pp 4467–4481 | Cite as

Engineered biosynthesis of cyclic lipopeptide locillomycins in surrogate host Bacillus velezensis FZB42 and derivative strains enhance antibacterial activity

  • Chuping LuoEmail author
  • Yongxing Chen
  • Xuehui Liu
  • Xiaohua Wang
  • Xiaoyu Wang
  • Xiangqian Li
  • Yuping Zhao
  • Lihui WeiEmail author
Applied genetics and molecular biotechnology


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.


Cyclic 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.

Funding information

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.

Ethical statements

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Ahimou F, Jacques P, Deleu M (2000) Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity. Enzyme Microb Tec 27:749–754CrossRefGoogle Scholar
  2. Arregui JR, Kovvasu SP, Betageri GV (2018) Daptomycin proliposomes for oral delivery: formulation, characterization, and in vivo pharmacokinetics. AAPS PharmSciTech 19:1802–1809. Epub 2018 Apr 3CrossRefGoogle Scholar
  3. Bais HP, Fall R, Vivanco JM (2004) Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol 134:307–319CrossRefGoogle Scholar
  4. Ballantine RD, Li YX, Qian PY, Cochrane SA (2018) Rational design of new cyclic analogues of the antimicrobial lipopeptide tridecaptin A1. Chem Commun (Camb) 54:10634–10637. Epub 2018 Sep 4CrossRefGoogle Scholar
  5. Borisova SA, Circello BT, Zhang JK, van der Donk WA, Metcalf WW (2010) Biosynthesis of rhizocticins, antifungal phosphonate oligopeptides produced by Bacillus subtilis ATCC6633. Chem Biol 17:28–37. CrossRefGoogle Scholar
  6. Burgard C, Zaburannyi N, Maier J, Jenke-Kodama H, Luxenburger E, Bernauer HS, Wenzel SC (2017)Genomics-guided exploitation of lipopeptide diversity in Myxobacteria. ACS Chem Biol 12:779–786. CrossRefGoogle Scholar
  7. Choi S-K, Park S-Y, Kim R, Kim S-B, Lee C-H, Kim JF, Park S-H (2009) Identification of a polymyxin synthetase gene cluster of Paenibacillus polymyxa and heterologous expression of the gene in Bacillus subtilis. J Bacteriol 191:3350–3358CrossRefGoogle Scholar
  8. 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. Epub 2017 Mar 24CrossRefGoogle Scholar
  9. 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
  10. 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. CrossRefGoogle Scholar
  11. 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. Google Scholar
  12. Gao L, Guo J, Fan Y, Ma Z, Lu Z, Zhang C, Zhao H, Bie X (2018) Module and individual domain deletions of NRPS to produce plipastatin derivatives in Bacillus subtilis. Microb Cell Factories 17:84. CrossRefGoogle Scholar
  13. 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. Epub 2017 Sep 1CrossRefGoogle Scholar
  14. Han L-L, Shao H-H, Liu Y-C, Liu G, Xie C-Y, Cheng X-J, Wang H-Y, Tan X-M, Feng H (2017) Transcriptome profiling analysis reveals metabolic changes across various growth phases in Bacillus pumilus BA06. BMC Microbiol 17:156. CrossRefGoogle Scholar
  15. Henry G, Deleu M, Thonart P, Ongena M (2011) The bacterial lipopeptide surfactin targets the lipid fraction of the plant plasma membrane to trigger immune-related defence responses. Cell Microbiol 13:1824–1837CrossRefGoogle Scholar
  16. Herzner AM, Dischinger J, Szekat C, Josten M, Schmitz S (2011) Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42. PLoS One 6(7):e22389. CrossRefGoogle Scholar
  17. Koh J, Lin S, Beuerman RW, Liu S (2017) Recent advances in synthetic lipopeptides as anti-microbial agents: designs and synthetic approaches. Amino Acids 49:1653–1677. Epub 2017 Aug 19CrossRefGoogle Scholar
  18. Koumoutsi A, Chen X, Henne A, Liesegang H, Hitzeroth G, Franke P, Vater J, Borriss R (2004) Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus. J Bacteriol 186:1084–1096CrossRefGoogle Scholar
  19. Koumoutsi A, Chen XH, Vater J, Borriss R (2007) DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42. Appl Environ Microbiol 73:6953–6964. CrossRefGoogle Scholar
  20. Leclere V, Bechet M, Adam A, Guez J-S, Wathelet B, Ongena M, Thonart P, Gancel F, Chollet-Imbert M, Jacques P (2005) Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism’s antagonistic and biocontrol activities. Appl Environ Microbiol 71:4577–4584CrossRefGoogle Scholar
  21. Lohani CR, Taylor R, Palmer M, Taylor SD (2015) Solid-phase total synthesis of daptomycin and analogs. Org Lett 17:748–751. CrossRefGoogle Scholar
  22. Loiseau C, Schlusselhuber M, Bigot R, Bertaux J, Berjeaud JM, Verdon J (2015) Surfactin from Bacillus subtilis displays an unexpected anti-Legionella activity. Appl Microbiol Biotechnol 99:5083–5093. CrossRefGoogle Scholar
  23. 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. CrossRefGoogle Scholar
  24. Luo C, Liu X, Zhou H, Wang X (2015a) Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions. Appl Environ Microbiol 81:422–431. CrossRefGoogle Scholar
  25. Luo C, Liu X, Zhou X, Guo J, Truong J, Wang X, Zhou H, Li X, Chen Z (2015b) Unusual biosynthesis and structure of locillomycins from Bacillus subtilis 916. Appl Environ Microbiol 81:6601–6609. CrossRefGoogle Scholar
  26. Mandal SM, Barbosa AEAD, Franco OL (2013) Lipopeptides in microbial infection control: scope and reality for industry. Biotechnol Adv 31:338–345. CrossRefGoogle Scholar
  27. Meena KR, Kanwar SS (2015) Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics. Biomed Res Int 2015:1–9. CrossRefGoogle Scholar
  28. Molle V, Fujita M, Jensen ST, Eichenberger P, González-Pastor JE, Liu JS, Losick R (2003) The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50:1683–1701. CrossRefGoogle Scholar
  29. 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. CrossRefGoogle Scholar
  30. Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16:115–125CrossRefGoogle Scholar
  31. Park S-Y, Choi S, Kim J, Oh T-K, Park S-H (2012) Efficient production of polymyxin in the surrogate host Bacillus subtilis by introducing a foreign ectB gene and disrupting the abrB gene. Appl Environ Microbiol 78:4194–4199CrossRefGoogle Scholar
  32. Patel S, Ahmed S, Eswari JS (2015) Therapeutic cyclic lipopeptides mining from microbes: latest strides and hurdles. World J Microbiol Biotechnol 31:1177–1193. CrossRefGoogle Scholar
  33. Parker JB, Walsh CT (2013) Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin. Biochemistry 52 (5):889-901CrossRefGoogle Scholar
  34. Raaijmakers JM, de Bruijn I, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev 34:1037–1062. CrossRefGoogle Scholar
  35. Radek K, Gallo R (2007) Antimicrobial peptides: natural effectors of the innate immune system. Semin Immunopathol 29:27–43. CrossRefGoogle Scholar
  36. Robbel L, Marahiel MA (2010) Daptomycin, a bacterial lipopeptide synthesized by a nonribosomal machinery. J Biol Chem 285:27501–27508.
  37. 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
  38. Roongsawang N, Washio K, Morikawa M (2011) Diversity of nonribosomal peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants. Int J Mol Sci 12:141–172. CrossRefGoogle Scholar
  39. Schneider T, Müller A, Miess H, Gross H (2014) Cyclic lipopeptides as antibacterial agents—potent antibiotic activity mediated by intriguing mode of actions. Int J Med Microbiol 304:37–43. CrossRefGoogle Scholar
  40. Schwarzer D, Finking R, M a M (2003) Nonribosomal peptides: from genes to products. Nat Prod Rep 20:275–287. CrossRefGoogle Scholar
  41. Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol 56:845–857CrossRefGoogle Scholar
  42. Strauch MA, Bobay BG, Cavanagh J, Yao F, Wilson A, Le Breton Y (2007) Abh and AbrB control of Bacillus subtilis antimicrobial gene expression. J Bacteriol 189:7720–7732. CrossRefGoogle Scholar
  43. Strieker M, Marahiel MA (2009) The structural diversity of acidic lipopeptide antibiotics. ChemBioChem 10:607–616. CrossRefGoogle Scholar
  44. Sun H, Bie X, Lu F, Wu Y, Lu Z (2009) Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter. Can J Microbiol 55:1003–1006CrossRefGoogle Scholar
  45. Sun J, Qian S, Lu J, Liu Y, Lu FX, Bie X, Lu Z (2018) Knockout of rapC improves the bacillomycin D yield based on de novo genome sequencing of Bacillus amyloliquefaciens fmbJ. J Agric Food Chem 66:4422–4430. CrossRefGoogle Scholar
  46. Tsuge K, Ano T, Hirai M, Nakamura Y, Shoda M (1999) The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Chemother 43:2183–2192CrossRefGoogle Scholar
  47. Tsuge K, Ohata Y, Shoda M (2001) Gene yerP, involved in surfactin self-resistance in Bacillus subtilis. Antimicrob Agents Chemother 45:3566–3573. CrossRefGoogle Scholar
  48. Wu L, Wu H, Chen L, Lin L, Borriss R, Gao X (2015) Bacilysin overproduction in Bacillus amyloliquefaciens FZB42 markerless derivative strains FZBREP and FZBSPA enhances antibacterial activity. Appl Microbiol Biotechnol 99:4255–4263. CrossRefGoogle Scholar
  49. Wu L, W H, Qiao J, Gao X, Borriss R (2019) Novel routes for improving biocontrol activity of Bacillus based bioinoculants. Front Microbiol 6:1–13. Google Scholar
  50. Xu Z, Zhang R, Wang D, Qiu M, Feng H, Zhang N, Shen Q (2014) Enhanced control of cucumber wilt disease by Bacillus amyloliquefaciens SQR9 by altering the regulation of its DegU phosphorylation. Appl Environ Microbiol 80:2941–2950. CrossRefGoogle Scholar
  51. Zavascki AP, Goldani LZ, Li J, Nation RL (2007) Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother 60:1206–1215. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Life Science and Food EngineeringHuaiyin Institute of TechnologyHuaianChina
  2. 2.Institute of Plant ProtectionJiangsu Academy of Agricultural SciencesNanjingChina
  3. 3.Institute of BiophysicsChinese Academy of SciencesBeijingChina

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