原生质体融合构建高效产脂肽工程菌

芽孢杆菌因其可产生多种生理活性物质,在环境污染修复、生物防治、微生物采油等领域具有广阔的应用前景。莫哈韦芽孢杆菌Bacillus mojavensis JF-2和解淀粉芽孢杆菌B. amyloliquefaciens BQ-6是从油田筛选出的产脂肽类表面活性剂菌株, 但在微生物采油实际应用中受到氧气浓度、盐度及pH的限制。原生质体融合是改变微生物代谢功能的一种简便有效的方法,以上述两株芽孢杆菌为对象,利用4因素3水平正交试验来探索菌龄、溶菌酶浓度、酶解温度和酶解时间对原生体制备、再生的影响。此外,对两菌株进行了双亲灭活原生质体融合,通过筛选得到了一株工程菌HY-4,并对其进行了初步的评价。结果表明:菌龄、溶菌酶浓度和酶解时间显著影响芽孢杆菌原生质体制备率及再生率(P<0.05),且在溶菌酶处理前用生理盐水多次洗涤菌体细胞,可提高制备率。两种芽孢杆菌原生质体制备及再生的最优条件均为:菌龄7 h、溶菌酶浓度2.5 mg/ml、酶解时间30 min、酶解温度42 ℃。融合子HY-4的最高耐盐度为15%,可耐50 ℃高温,代谢产脂肽的pH范围为4.0~9.5,且在好氧及厌氧条件下均能够代谢产脂肽,在厌氧条件下生长迅猛(细胞干重>1.6 g/L)。综上所述,融合子HY-4具有较大的应用潜力,该研究为芽孢杆菌的遗传育种打下了方法学基础,并对驱油微生物菌种的选育具有指导意义。

Construction of Engineered Bacteria for Efficient Lipopeptide Production by Protoplast Fusion

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.