Breeding Of Epothilone High Producing Strains Of Sorangium Cellulosum By Genome Shuffling

Epothilone is a novel class of macrolide compounds fermented by Sorangiumcellulosum, which is of anti-tumor activity. Compared with the traditional anti-cancerdrug Taxol, epothilone not only has bertter antiproliferative activity, but also isdistinguished for its simple molecular structure, good water-solubility, lower toxicityand side effects, wide antitumor spectrum, and character of fermentable, etc, thusepothilone is an important anti-tumor drug with important development potential.However, beacause of the lack of awareness about the genetic background of theSprangium cellulosum, the inefficient genetic operation system function and the absenceof effective genetic markers and so on, the genetic alteration of the Sprangiumcellulosum and the heterologous expression of the epothilones both encountered greatdifficulties. So using conventional means to screen microorganisms is the primarymethod to improve the production of the epothilones at present.Genome shuffling which was put forward in recent years is a simple and efficientstrain improvement way, it is based on several rounds of recursive protolast fusiontechnology and combine fine properties of different parents, so mutant strains could beobtained with evidently improved characters in a short time, it has been proved in manyindustrial strain screening now. In this article, screening of strain was started from wildS.cellulosum stain So0157-2to large numbers of fusants and obtain strain which yield issignificantly increased through pH, tolerance breeding of precursors and products,dispersion acclimation genome shuffling tolerance and high-throughput screeningmethod.Main work is as follows:1. The high-producing strain Pe107through breeding of pH and tolerance to theprecursors and product was obtained. The yield of epothilone A and B of strain Pe107are respectively improved to227%and210%, and the expected characters of thetolerance to high density precursors and product has reached.2. Because of exsiting agglomerate growth characteristics in liquid culture,Sorangium cellulosum is not conducive to fermentation. Through the supernatantsubcultured domestication of the pH tolerance mutations have dispersed growth thestrain L4-29dispersed growth trend type. Although in the fermentation process bacterialquantum and production decreased significantly, the fermentation liquid is almost turbid homogeneous liquid. The degree of dispersion than the wild type strain is about9timesincreased trends obtained dispersed growth types strain L4-29.3. Then the conditions of preparation, regeneration, inactivation, and fusionconditions of protoplast were discussed, Sorangium cellulosum Protoplast preparationconditions is taking seeds of three days, washing2times by PBS, adding3g/L lysozymeenzymatic90min after15%of EDTA pretreatment for30minutes; regenerationconditions is washing residual enzyme solution three times by14%mannitol hypertonic,and then M26liquid medium composition rejuventing containing10%hypertonic after24hours, including10%of the water suspended bacteria and then dumping agarhypertonic of ingredients based on VY/2regeneration solid culture; lost activitiesmechanical condition30W ultraviolet irradiation distance in5cm for3minutes55℃ofwater to take a shower10minutes; fusion conditions is30℃water bathing for5min afteradding30%PEG.4. The strain Pe107with high production and dispersion growth pattern strainL4-29were subjected to genome shuffling breeding by using the protoplast fusioncondition. Fusion strain Gc16was got with high production of epothilone A and B, theproduction of the fusant was improved to117.9%and117.1%compared to the parents,and was improved to267.6%and246.2%compared to the starting strain.5. The feasibility of the method for screening the strain that produces higherepothilone B was analyzed which based on the method that combined micro-cultivationin96-well microtiter plates and microplate spectrograph. The method is suitable for thehigh throughput screening.96-well microtiter plates was used in solid fermentation totraining the strain. OD₂49of those extractions were detected by microplate spectrometerand the yields of epothilone B were detected by HPLC. SPSS analysis showed that theOD₂49was in good correspondence to the epothilone B content, and the top20%strainsin OD₂49could be used in rescreening. Further experiment showed that the epothilone Bcontent and OD249of the extract had a good stability in24hours.