利用特比萘芬抗性筛选赤霉素高产菌株及相关发酵特性的研究

Screening and identification of high gibberellin-producing strain from terbinafine resistant mutants

摘要:

【目的】诱变、筛选赤霉素高产菌株，并明确该菌遗传稳定性及部分发酵特性。【方法】利用亚硝基胍、60Co-γ射线以及复合诱变的方法，结合抑真菌剂-特比萘芬抗性筛选，定向选育赤霉素高产突变株；通过琼脂斜面传代实验确定遗传稳定性；发酵罐培养了解其部分发酵性状。【结果】藤仓赤霉菌单细胞悬液先后经过终浓度为300 mg/L亚硝基胍30 min和60Co-γ射线300戈瑞下复合诱变，得到抗120 μg/L特比萘芬的赤霉菌突变株。摇瓶复筛，确定赤霉素突变菌株的赤霉素合成能力，其中ZNL13-3菌产赤霉素效价为2 215±35 mg/L，较之诱变前发酵效价平均提高了11.87%。ZNL13-3菌株经连续15次试管斜面传代考察，菌株赤霉素合成的稳定性较好，能维持原发菌株93.2%，具有良好的遗传稳定性和生产应用前景。5 L发酵罐培养，比较菌丝浓度和产物浓度随发酵时间变化趋势，ZNL13-3菌比生产速率优于原发菌株。【结论】赤霉菌抗特比萘芬的变异特性与赤霉素高产之间存在某种对应关系，为进一步定向优化筛选优良菌株提供参考。

Abstract:

[Objective] The high gibberellins-producing strains were screened and identified. Meanwhile, studies on characterization of genetic stability and fermentation in the bioreactor were also carried out. [Methods] Used the NTG, 60Co-γ rays and composite mutagenesis methods, high-yield mutant strains were isolated directionally in the medium including the antifungal agents-terbinafine. The genetic stability of mutant strain was determined by the several generations on the agar slants. The part of its fermentation characteristics were studied in the fermentation tank. [Results] The strains suspension was treated with the mutagens including a final NTG concentration 200 mg/L for 30 min, followed by 60Co gamma ray (300 Gy). The mutant strains were isolated on the PDA plate containing 120 μg/L terbinafine. After further shaking flask experiments, potentials of the mutant strains were determined. The fermentation titer of strain ZNL13-3 was 2 245±35 mg/L, increased 11.87% compared with the primary strains. The ZNL13-3 has a good genetic stability and value of application. Fermentation performance of ZNL13-3 is better than the primary strains, compared with the time trends of mycelia concentration and gibberellin acid concentration in the 5 L bioreactor. [Conclusion] The high-yield strains of gibberellin acid could be found and its ability to produce gibberellin acid could be affected by the terbinafine resistance.