A Rapid and Specific Method to Screen Epothilone High-Producing Strain with Spectrometry and its Application
Epothilones are a kind of poliketide macrolide with strong stabilizing activities on polymerized microtubules which mimicked taxol. Microbial fermentation of Sorangium cellulosum is still the main way to produce epothilones with strain continuously improv
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A Rapid and Specific Method to Screen Epothilone High-Producing Strain with Spectrometry and its Application Lin Zhao, Xin Sun, Yawei Li, Haiyan Gao, Qiang Ren, Yongwei Hao, Song Zhang and Xinli Liu
Abstract Epothilones are a kind of poliketide macrolide with strong stabilizing activities on polymerized microtubules which mimicked taxol. Microbial fermentation of Sorangium cellulosum is still the main way to produce epothilones with strain continuously improving by mutation. In general, yields of epothilones were detected by HPLC or LC–MS, which were time-consuming and not suited for high-throughput screening. In this paper, we described an efficient high-throughput method for epothilones high-producing strains with 96-well micro titer plate spectrometry combined with CCl4 extracting. In this method, the purity of epothilones was 22 times higher than methanol extract. So the OD249 was much less disturbed by the impurities. Stabilization experiment and SPSS analysis of the relationship between the yields of epothilones detected by HPLC and the OD249 of the CCl4 extracts of 192 mutated strains showed that our method was practical, accurate, and much more efficient.
Keywords Epothilones Sorangium cellulosum Rapid screening Spectrometry
7.1 Introduction Epothilones are a kind of poliketide macrolide with strong stabilizing activities on polymerized microtubules which mimicked taxol [1, 2] (Fig. 7.1). They are biosynthesized by a NRPS/PKS hybrid gene cluster in a stepwise polymerization
Zhao Lin and Sun Xin contributed equally to this work. L. Zhao X. Sun Y. Li H. Gao Q. Ren Y. Hao S. Zhang X. Liu (&) Shandong Provincial Key Laboratory of Microbial Engineering, Qilu University of Technology, Jinan 250353, People’s Republic of China e-mail: [email protected]
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 249, DOI: 10.1007/978-3-642-37916-1_7, Ó Springer-Verlag Berlin Heidelberg 2014
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sequence in the genome of Sorangium cellulosum. Until their anticancer bioactivity was reported, more and more following extensive studies on epothilones in various aspects were performed, and many synthetic and semi-synthetic epothilone analogues have been produced to further improve the adverse effect profile and to maximize the antitumor properties [3, 4]. Several epothilones and chemically modified derivatives of epothilones are being used in clinical anticancer trials [5–7], and ixabepilone (16-aza-epothilone B, developed by Bristol-Myers Squibb) was authorized for clinical use by the Food and Drug Administration of the United States in 2007 [8]. For lacking of efficient molecular techniques, genetic engineering is hardly performed in Sorangium cellulosum. Microbial fermentation is still the main way to produce epothilones with strain continuously improving by mutation. In general, yields of epothilone were detected by HPLC or LC–MS [9–11], which were timeconsuming and not suited f
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