通过两种糖多孢菌属菌株原生质体融合提高多杀菌素产量研究

Study on the increase of spinosad yields by protoplast fusion of two kinds of Saccharopolyspora strains

多杀菌素是由放线菌刺糖多孢菌产生的一种新型绿色环保杀虫剂,兼具化学农药的高效性与生物农药的安全性。本研究通过将红色糖多孢菌和刺糖多孢菌两种菌株做原生质体融合探究来得到高产多杀菌素的菌株。首先构建了不产红霉素的红色糖多孢菌工程菌株,然后将其与刺糖多孢菌做原生质体融合,经加有抗生素平板培养基及发酵液HPLC分析筛选,得到了产多杀菌素的4株融合菌株。基于4株融合菌株多杀菌素产量都不及起始菌株高,接着采用不加抗生素直接筛选的方法,成功筛选到若干高产多杀菌素的融合菌株,其中菌株B-2多杀菌素产量提高最为明显,较原始菌株增加了331%,并用质谱分析做了进一步的验证。融合菌株B-2后续传代培养多杀菌素产量稳定,表明遗传稳定性较好。本文对红色糖多孢菌和刺糖多孢菌原生质体融合进行了探究并且成功提高了多杀菌素的产量,这为具有优良发酵特性的红色糖多孢菌和刺糖多孢菌融合菌株的构建提供了重要基础。

Spinosad,produced by Saccharopolyspora spinosa is a new kind of green pesticides with efficacy of chemical pesticides and security of biological ones.This study tries to construct a bacterial strain with high yield of spinosad by protoplast fusion between Saccharopolyspora spinosa and Saccharopolyspora erythraea.A strain which did not produce erythromycins was first constructed and protoplast fusion between it and Saccharopolyspora spinosa was done then.After screening by culture of antibiotic plate medium and HPLC analysis of fermentation broth,four fused strains which could produce spinosyns were acquired.Because the yield of spinosad of all the four fused strains was lower than the original ones,direct screening without antibiotics was done then and a number of fused strains with high-yield spinosad were acquired successfully.Strain B-2,one of fused strains,had the highest production of spinosad with a increase of 331%compared with the original ones.Further confirmation for spinosyns produced by fused strain B-2 were obtained by analysis of mass spectrometry.The production of spinosad of B-2 was stable in the subsequent subculture,which indicated the genetic stability of B-2 was good.This research is a report on enhanced production of spinosad by protoplast fusion between Saccharopolyspora spinosa and Saccharopolyspora erythraea and will provide the important basis for constructing the fused strains with excellent features from Saccharopolyspora spinosa and Saccharopolyspora erythraea.