Abstract: A Streptomyces strain with high production of cycloheximide was chosen for a systematic genetic manipulation study.Conjugation,mycelium electroporation and PEG mediate protoplast transformation were used for the introduction of foreign genes.Seven different kinds of medium were tested to optimize fermentation condition and HPLC were used to analyze the fermentaiton products.λ-RED mediate PCR-targeting was used to knockout a possible PKS fragment which is supposed to be a key gene for CHX biosynthesis.As a result,modified conjugation using fresh spores was proved was be a valid method for introducing foreign genes into this strain,with relatively high conversion efficiency (10-6). Fermentation and HPLC condition were optimized.By using the genetic manipulation system we constructed,a PKS gene which is responsible to the biosynthesis of cycloheximide was found.The study provided a good example for exploring the genetic manipulation on streptomyces and made a solid foundation for obtaining novel compounds and improving the compounds production by optimizing genetic engineering of the strain.