Studies On The Fermentation Conditions Of Epothilone Engineering Bacteria

Epothilones, produced by Sorangium cellulosum, is a macrolide natural product with high antitumor activity and thus has commerlized as an antitumor drug. However,&cellulosum grows sloely, therefore, genetic engineering the biosynthetic gene cluster for heterologous expression and optimization fermentation conditions become effective way to improve the yield of epothilone.Medium optimization is a very important method to improve the titer of fermented product on the study of fermentation process. LC/MS/MS analysis the Burkholderia containing the epothilone gene cluster showed that epothilone was produced in heterologous host successfully. Then the fermentation conditions were studied. By tracking the epothilone production of the engineered strain in different fermentation conditions we obtained the optimal conditions for producing epothilone. This work provide a foundation for improve epothilone production.In this study, the engineering bacterium was cultivated in six different mediums, the bacteria concentration, cytotoxicity of the bacteria, and the epothilone production were analyzed and compared.The results show that the bacteria concentration of the3rd medium is the best candidate in the six medium. The maximum value of OD600reached12.58, it is3times to the2nd medium and6times to No.1,4,5,6medium of bacteria concentration at the same time.The maximum absorbance of No.1,4,5,6medium does not exceed2, the lowest bacteria concentration of medium is the5th medium, the maximum absorbance is1.2. the cell growth inhibition rate of the extracts of engineering strain cultured by1-6culture medium were57.94%、83.81%、65.36%、1.87%、7.74%、57.76%. Combined with the six kinds of medium growth curve, showing the positive correlation between bacteria concentration and epothilone production is not obvious.Precious researches showed that the supply of the precursor played an important role in the fermentation process. Adding the appropriate precursor can effectively improve the production. For further improving the epothilone production, we used single factor analysis to study six possible precursor substances. The results show that all precursor substances when fed in proper concentration could promote epothilone production, and factor B (sodium propionate)> D (serine)> F (trace element)> C (sodium citrate)> A (sodium acetate)> E (cysteine). Adding5,10and20μg/ml of sodium propionate, cell growth inhibition rate of engineered bacteria increased from57.94%to89.18%,92.8%,91.21%respectively, and adding5ug/ml of serine, cell growth inhibition rate raised from57.94%to92.55%. But excessive cysteine and sodium acetate have inhibitory effect on biosynthesis of the cytotoxin products of engineered bacteria. Adding80μg/ml of sodium acetate, cell growth inhibition rate fell from57.94%to56.05%, add10μg/ml cysteine, cell growth inhibition rate of engineering bacteria droped from57.94%to14.57%.Transcription and expression of the target gene were regulated strictly by promoters. Using strong promoters may facilitate transcription of the target gene significantly. In this study, through a series of genetic engineering methods such as enzyme digestion and Red/ET technology et al, two plasmids:pR6K-Amp-H.a-f-PTet-H.a-r and pR6K-H.a-f-PBAD-H.a-r.The plasmids containing upstream/downstream homology arms, R6Kori and strong promoter PTet or PBAD were constucted. The two plasmids were expected to transfomed into engineered bacteria, and replace its original promoter forpromoting epothilone biosynthesis.The constuction of strong promoter plasmids provided the basis for promoting the efficient production of epothilones in engineered bacteria.