Skip to main content
Log in

Improvement of lichenysin production in Bacillus licheniformis by replacement of native promoter of lichenysin biosynthesis operon and medium optimization

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript


Lichenysin is a biodegradable surfactant with huge potential for recovering crude oil from the oil reservoir. The current production of lichenysin is made through fermentation from wild strain of Bacillus licheniformis, which is limited by low yield. The aim of this work was to improve lichenysin-producing capability of a wide strain B. licheniformis WX-02. Lichenysin produced from WX-02 was first extracted, purified, and identified. Through the substitution of the promoter of lichenysin biosynthesis operon, the mutants B. licheniformis WX02-P43lch, WX02-Pxyllch, and WX02-Psrflch were constructed with the constitutive promoter (P43), the xylose-inducible promoter (P xyl ), and the surfactin operon promoter (P srf ), respectively. A consistent change trend was observed between lichenysin production and lchAA gene transcription, confirming the strength of the promoters as an important factor for lichenysin synthesis. Among the three mutants, WX02-Psrflch produced the highest lichenysin yield. The production by the mutant WX02-Psrflch was further improved with the optimization of the major medium components including glucose, NH4NO3, and Na2HPO4/KH2PO4. Under 30 g/L glucose, 5 g/L NH4NO3, and 80 mM/60 mM Na2HPO4/KH2PO4, the strain WX02-Psrflch produced 2,149 mg/L lichenysin, a 16.8-fold improvement compared to that of wild strain WX-02.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others


  • Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, Fracchia L, Smyth TJ, Marchant R (2010) Microbial biosurfactants production, applications and future potential. Appl Microbiol Biot 87(2):427–44. doi:10.1007/s00253-010-2589-0

    Article  CAS  Google Scholar 

  • Duan YX, Chen T, Chen X, Zhao XM (2010) Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis. Appl Microbiol Biot 85(6):1907–14. doi:10.1007/s00253-009-2247-6

    Article  CAS  Google Scholar 

  • Duitman EH, Wyczawski D, Boven LG, Venema G, Kuipers OP, Hamoen LW (2007) Novel methods for genetic transformation of natural Bacillus subtilis isolates used to study the regulation of the mycosubtilin and surfactin synthetases. Appl Environ Microb 73(11):3490–3496

    Article  CAS  Google Scholar 

  • Fickers P, Guez J-S, Damblon C, Leclère V, Béchet M, Jacques P, Joris B (2009) High-level biosynthesis of the anteiso-C17 isoform of the antibiotic mycosubtilin in Bacillus subtilis and characterization of its candidacidal activity. Appl Environ Microb 75(13):4636–4640

    Article  CAS  Google Scholar 

  • Gat O, Inbar I, Aloni-Grinstein R, Zahavy E, Kronman C, Mendelson I, Cohen S, Velan B, Shafferman A (2003) Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines. Infect Immun 71(2):801–813. doi:10.1128/iai.71.2.801-813.2003

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Grangemard I, Wallach J, Maget-Dana R, Peypoux F (2001) Lichenysin. Appl Biochem Biotech 90(3):199–210

    Article  CAS  Google Scholar 

  • Jacques P (2011) Surfactin and other lipopeptides from Bacillus spp. In: Soberón-Chávez G (ed) Biosurfactants. Microbiology Monographs, vol 20. Springer Berlin Heidelberg, pp 57-91

  • Joshi S, Yadav S, Desai AJ (2007) Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51. J Microbiol Biotechn 17(2):313–319

    CAS  Google Scholar 

  • Joshi S, Yadav S, Desai AJ (2008) Application of response-surface methodology to evaluate the optimum medium components for the enhanced production of lichenysin by Bacillus licheniformis R2. Biochem Eng J 41(2):122–127

    Article  CAS  Google Scholar 

  • Jung J, Yu KO, Ramzi AB, Choe SH, Kim SW, Han SO (2012) Improvement of surfactin production in Bacillus subtilis using synthetic wastewater by overexpression of specific extracellular signaling peptides, comX and phrC. Biotechnol Bioeng 109(9):2349–56. doi:10.1002/bit.24524

    Article  PubMed  CAS  Google Scholar 

  • Kim JH, Hwang BY, Roh J, Lee JK, Kim K, Wong SL, Yun H, Lee SG, Kim BG (2008) Comparison of P aprE , P amyE , and P P43 promoter strength for β-galactosidase and staphylokinase expression in Bacillus subtilis. Biotechnol Bioproc E 13(3):313–318

    Article  CAS  Google Scholar 

  • Kracht M, Rokos H, Ozel M, Kowall M, Pauli G, Vater J (1999) Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. J Antibiot 52(7):613–619

    Article  PubMed  CAS  Google Scholar 

  • Li Y-M, Haddad NIA, Yang S-Z, Mu B-Z (2008) Variants of lipopeptides produced by Bacillus licheniformis HSN221 in different medium components evaluated by a rapid method ESI-MS. Int J Pept Res Ther 14(3):229–235. doi:10.1007/s10989-008-9137-0

    Article  CAS  Google Scholar 

  • Lin S-C, Lin K-G, Lo C-C, Lin Y-M (1998) Enhanced biosurfactant production by a Bacillus licheniformis mutant. Enzyme Microb Tech 23(3–4):267-273 doi:

  • Liu JF, Yang J, Yang SZ, Ye RQ, Mu BZ (2012a) Effects of different amino acids in culture media on surfactin variants produced by Bacillus subtilis TD7. Appl Biochem Biotech 166(8):2091–100. doi:10.1007/s12010-012-9636-5

    Article  CAS  Google Scholar 

  • Liu X, Ren B, Gao H, Liu M, Dai H, Song F, Yu Z, Wang S, Hu J, Kokare CR (2012b) Optimization for the production of surfactin with a new synergistic antifungal activity. PLoS One 7(5):e34430

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Madslien EH, Rønning HT, Lindbäck T, Hassel B, Andersson MA, Granum PE (2013) Lichenysin is produced by most Bacillus licheniformis strains. J Appl Microbiol 115(4):1068–1080. doi:10.1111/jam.12299

    PubMed  CAS  Google Scholar 

  • Masuda A, Dohmae N (2012) Amino acid analysis of sub-picomolar amounts of proteins by precolumn fluorescence derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Biosci Trends 5(6):231–238. doi:10.5582/bst.2011.v5.6.231

    Article  Google Scholar 

  • Mikkola R, Kolari M, Andersson MA, Helin J, Salkinoja-Salonen MS (2000) Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis. Eur J Biochem 267(13):4068–4074

    Article  PubMed  CAS  Google Scholar 

  • Nerurkar AS (2010) Structural and molecular characteristics of lichenysin and its relationship with surface activity. Adv Exp Med Biol 672:304–315

    Article  PubMed  CAS  Google Scholar 

  • Qi G, Kang Y, Li L, Xiao A, Zhang S, Wen Z, Xu D, Chen S (2014) Deletion of meso-2, 3-butanediol dehydrogenase gene budC for enhanced D-2, 3-butanediol production in Bacillus licheniformis. Biotechnol Biofuels 7(1):16

    Article  PubMed  PubMed Central  Google Scholar 

  • Radha S, Gunasekaran P (2008) Sustained expression of keratinase gene under P xylA and P amyL promoters in the recombinant Bacillus megaterium MS941. Bioresource Technol 99(13):5528–5537

    Article  CAS  Google Scholar 

  • Rangarajan V, Dhanarajan G, Kumar R, Sen R, Mandal M (2012) Time-dependent dosing of Fe2+ for improved lipopeptide production by marine Bacillus megaterium. J Chem Technol Biot 87(12):1661–1669. doi:10.1002/jctb.3814

    Article  CAS  Google Scholar 

  • Razafindralambo H, Popineau Y, Deleu M, Hbid C, Jacques P, Thonart P, Paquot M (1998) Foaming properties of lipopeptides produced by Bacillus subtilis: effect of lipid and peptide structural attributes. J Agr Food Chem 46(3):911–916

    Article  CAS  Google Scholar 

  • Satpute SK, Bhuyan SS, Pardesi KR, Mujumdar SS, Dhakephalkar PK, Shete AM, Chopade BA (2010) Molecular genetics of biosurfactant synthesis in microorganisms. Adv Exp Med Biol 672:14–41

    Article  PubMed  CAS  Google Scholar 

  • Tao X, Liu Y, Wang Y, Qiu Y, Lin J, Zhao A, Su M, Jia W (2008) GC-MS with ethyl chloroformate derivatization for comprehensive analysis of metabolites in serum and its application to human uremia. Anal Bioanal Chem 391(8):2881–2889

    Article  PubMed  CAS  Google Scholar 

  • Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biot 72(2):211–22. doi:10.1007/s00253-006-0465-8

    Article  CAS  Google Scholar 

  • Wei X, Ji Z, Chen S (2010) Isolation of halotolerant Bacillus licheniformis WX-02 and regulatory effects of sodium chloride on yield and molecular sizes of poly-γ-glutamic acid. Appl Biochem Biotech 160(5):1332–1340

    Article  CAS  Google Scholar 

  • Wei X, Tian G, Ji Z, Chen S (2014) A new strategy for enhancement of poly-γ-glutamic acid production by multiple physicochemical stresses in Bacillus licheniformis. J Chem Technol Biot. doi:10.1002/jctb.4362

  • Yakimov MM, Timmis KN, Wray V, Fredrickson HL (1995) Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. Appl Environ Microb 61(5):1706–1713

    CAS  Google Scholar 

  • Yakimov MM, Fredrickson HL, Timmis KN (1996) Effect of heterogeneity of hydrophobic moieties on surface activity of lichenysin A, a lipopeptide biosurfactant from Bacillus licheniformis BAS50. Biotechnol Appl Bioc 23(1):13–18. doi:10.1111/j.1470-8744.1996.tb00358.x

    CAS  Google Scholar 

  • Yakimov MM, Giuliano L, Timmis KN, Golyshin PN (2000) Recombinant acylheptapeptide lichenysin: high level of production by Bacillus subtilis cells. J Mol Microb Biotech 2(2):217–224

    CAS  Google Scholar 

  • Yang M, Zhang W, Ji S, Cao P, Chen Y, Zhao X (2013) Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system. PLoS One 8(2):e56321

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Yangtse W, Zhou Y, Lei Y, Qiu Y, Wei X, Ji Z, Qi G, Yong Y, Chen L, Chen S (2012) Genome sequence of Bacillus licheniformis WX-02. J Bacteriol 194(13):3561–3562

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Youssef NH, Duncan KE, McInerney MJ (2005) Importance of 3-hydroxy fatty acid composition of lipopeptides for biosurfactant activity. Appl Environ Microbiol 71(12):7690–5. doi:10.1128/AEM.71.12.7690-7695.2005

    Article  PubMed  CAS  PubMed Central  Google Scholar 

Download references


This work was supported by rural areas of the national science and technology plan in the 12th 5-year plan of China (No. 2013AA102801-52) and the National Natural Science Foundation of China (Grant No. 31170046).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Shouwen Chen.

Electronic supplementary material

Below is the link to the electronic supplementary material.


(PDF 1122 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qiu, Y., Xiao, F., Wei, X. et al. Improvement of lichenysin production in Bacillus licheniformis by replacement of native promoter of lichenysin biosynthesis operon and medium optimization. Appl Microbiol Biotechnol 98, 8895–8903 (2014).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: