EctB Gene And Culture Conditions Of Regulation To Effect The Production Of Polymyxin In Paenibacillus Polymyxa

In this research, the recombinant strain P.polymyxa 1-8/pMD516-ectB was constructed to study the influence of regulating ectB copy on polymyxin production. The medium components were optimized via several experiments such as single factorial, Plackett-Burman, steepest ascent and response surface for improvement of polymyxin production by Paenibacillus polymyxa 1-8. Moreover, the cultivation of strain was conducted in shake flask using the optimal medium, and the effect of some culture parameters on polymyxin production was investigated. Accordingly, some regulation strategies were proposed and carried out in 5 L fermentor.(1) Two PCR primers were designed and synthesized to amplifying P.polymyxa ectB gene from genome of P.polymyxa 1-8. The ectB gene was digested and inserted into pMD516 plasmid by using EcoR I and Xho I in order to obtain the recombinant plasmid pMD516-ectB. Both recombinationt(pMD516-ectB) and contral plasmid(pMD516) were transformed into P.polymyxa 1-8 respectively by electroporation for acquirement of a recombinant strain(P.polymyxa 1-8/pMD516-ectB) and a contral strain(P.polymyxa 1-8/pMD516). The titers of polymyxin produced by P.polymyxa 1-8/pMD516-ectB and P.polymyxa 1-8/pMD516 were compared, and it was found that a higher titer of 36,100 U/mL was achieved in the contral strain but a lower in the recombinant strain(28,600 U/mL). The results showed that increase copy number of ectB gene for the strain P.polymyxa 1-8 could not enhance the polymyxin production.(2) The culture medium was optimized when P.polymyxa 1-8 was cultivated in shake flask. Soluble starch and zinc sulfate were determined to be the most suitable carbon source and inorganic salt respectively, by the single factorial experiment. Then, the significant factors were confirmed by Plackett-Burman design, including organic nitrogen source, inorganic nitrogen and calcium carbonate. The steepest ascent experiment was adopted to define the center point of response surface, and the point was 48 g/L mixed soybean meal and flour, 29 g/L ammonium sulfate, 8 g/L calcium carbonate. The optimal medium components were predicted by the Box-Behnken experiment, and listed as follow: soluble starch 25 g/L, mixed defatted soybean meal and flour 51 g/L, ammonium sulfate 29.3 g/L, zinc sulfate 0.1 g/L, calcium carbonate 7.83 g/L, dipotassium hydrogenphosphate 0.77 g/L. When P.polymyxa 1-8 was culured in the optimal medium, the production of polymyxin reached to 46,500 U/mL, which is a 31.4% increment compared to that using the medium before optimization.(3) When fermenting P.polymyxa 1-8 in shake flask,the inoculum of 7% and shake speed of 140 r/min were determined to be the optimum condition for polymyxin production. Aimed to explore the optimum environment parameters during the period of polymyxin production, the different temperatures and pH values in this period were searched, and it turned out that keeping 30℃ and pH 5-6 in this period were beneficial for polymyxin production. Accoding to the results above, the regulation strategies of fermentation process were proposed, and carried out in a 5 L fermentor. The results indicated that a high titer of 66,400 U/mL was achieved, when temperature was maintained at 30℃ during the fermentation process and pH was regulated between 5 to 6 during the polymyxin production period, with 4 times feeding of starch hydrolysate digested by amylase at 23, 31, 39 and 43 h respectively. This titer obtained in fermentor under the optimum condition was 42.8% higher than that in shake flask.