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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2014, Vol. 34 Issue (11): 76-84    DOI: 10.13523/j.cb.20141111
    
Construction of Engineered Bacteria for Efficient Lipopeptide Production by Protoplast Fusion
LIANG Xiao-long1,2, ZHAO Feng3, SHI Rong-jiu1, ZHOU Ji-dong1,2, HAN Si-qin1, ZHANG Ying1
1. Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China;
3. State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China
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Abstract  

Bacillus can produce a variety of physiologically active substances and has broad application potential in environmental remediation, biological control, oil recovery and other fields. Bacillus mojavensis JF-2 and B. amyloliquefaciens BQ-6 are high-yielding strains of lipopeptide, screened from oil field, but their practical application in microbial enhanced oil recovery (MEOR) is restricted by oxygen concentration, salt concentration and pH. It is a simple and efficient approach to transform microbial metabolic functions by protoplast fusion. In this research, the above two Bacillus strains were taken as the research objects, and a L9(34) orthogonal experiment was conducted to explore the optimal conditions for protoplast preparation and regeneration. In addition, engineered bacteria were constructed by inactivated protoplast fusion. The results showed that cell age, lysozyme concentration and enzymolysis time significantly affected the rate of Bacillus protoplast preparation and regeneration rates (P<0.05), and the preparation rate was improved markedly when the Bacillus cells were repeatedly washed with sterile normal saline before enzymolysis. The optimum conditions for protoplast formation and regeneration of both Bacillus strains were: 7-hour cell age, 2.5 mg/ml lysozyme concentration, 30-minute enzymolysis, and 42 ℃ of lysozyme temperature. Fusant HY-4, salt tolerance of 15% and pH range of 4.0 to 9.0 for lipopeptide production, was capable of producing metabolic lipopeptide under both aerobic and anaerobic conditions, and it exhibited a rapid anaerobic growth (dry cell weight>1.6 g/L). In summary, fusant HY-4 has a great application potential, and this research laid a foundation for Bacillus genetic breeding methodology, providing a reference for breeding oil-flooding microorganisms.

Key words： Bacillus      Protoplast      Orthogonal experimental design      MEOR      Genetic breeding     
Received: 23 September 2014      Published: 25 November 2014
ZTFLH:  Q939  
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Cite this article:

LIANG Xiao-long, ZHAO Feng, SHI Rong-jiu, ZHOU Ji-dong, HAN Si-qin, ZHANG Ying. Construction of Engineered Bacteria for Efficient Lipopeptide Production by Protoplast Fusion. China Biotechnology, 2014, 34(11): 76-84.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20141111     OR     https://manu60.magtech.com.cn/biotech/Y2014/V34/I11/76


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