PDF(Pubmed)
Abstract：
Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.
摘要：
生物表面活性剂具有显著特点,如环境友好，安全性高,和优异的生物降解性。表面活性剂是由枯草芽孢杆菌生产的最著名的生物表面活性剂之一。因为生物表面活性剂的生物合成途径，比如surfactin,是复杂的，诱变是开发高产菌株的典型代谢工程方法的有用替代方法。因此,对于来源于突变文库的高产量菌株,需要高通量和精确的筛选方法。血琼脂裂解法,利用生物表面活性剂的溶血活性，是确定其浓度的一种方法。该方法包括将微生物细胞接种到含有血液的琼脂平板上，基于每个菌落周围形成的溶血区的大小来评估生物表面活性剂的产生。血琼脂裂解方法的挑战包括低实验再现性和缺乏建立的高通量筛选方案。因此,在这项研究中，我们研究了接种程序和培养基成分对溶血区形成的影响。我们还开发了一个使用机器人技术评估菌落数量的工作流程。结果显示,通过以适当的间隔排列菌落,利用图像分析软件测量菌落和溶血环的面积,可以准确比较几个菌落之间的溶血活性。尽管使用血琼脂裂解法进行筛选仅限于具有溶血活性的表面活性剂,相信通过考虑从这项研究中获得的见解，有助于准确筛选高产菌株。
