Research On Fermentation Engineering Of Saccharopolyspora Erythraea WB And Gene Engineering Bacterias

Saccharopolyspora erythraea is actinomycete producing erythromycin in industry. Erythromycin is a broad-spectrum 14- membered macrolide antibiotics.It has been widely used in the treatment of gram positive bacterial infections. In recent years, erythromycin industry developed rapidly in our country. Improvement and the improvement of the production of erythromycin group is mainly depends on the strain of their own fermentation ability, fermentation technology and fermentation ability. With the increasing demand of erythromycin Market, the pace of erythromycin high-yielding strain screening is also in constant speed. Screening of high yield strains of genetically engineered is a simple operation method.TetR family transcriptional regulator is one of the most common family of nuclear transcriptional regulation named after the resistance to tetracycline repressor protein. SACE₃446 gene is a negative regulator of erythromycin biosynthesis gene exists in Saccharopolyspora erythraea while SACE₇301 is a Saccharopolyspora erythraea TetR family transcriptional regulator. It can positively regulate the production of erythromycin and is a positive regulation gene.Taking the Saccharopolyspora erythraea and the secondary metabolites erythromycin as the object, the study, using the high genetic engineering strain which is obtained through constructing and screening, puts its emphasis on its productivity via the shake flask fermentation experiment. Taking 5L fermentor incubation methods as the major incubation methods, it was analyzed and verified that various factors, including temperature, pH value, stirring paddle, revolving speed, inoculum concentration, seed age and so on, will bring effects on growth and metabolism during the fermentation of Saccharopolyspora erythraea WB bacterial strain. By using single factor experiment to determine the above three factors including temperature, inoculation amount, seed age Box-Benhnken design. And in the design of Box-Benhnken, the range of temperature, inoculation amount, seed age can be determined. On the above basis, the optimization of response surface test can be done, and the range of the above three factors will be further determined. Finally, a small fermentation, which is more suitable for Saccharopolyspora erythraea strain WB, has been summarized and is used to do small fermentation experiments about genetic engineering strain WB/3446 and WB/3* 7301; the similarities and differences between small fermentation of genetically engineered strains and that of original strains have been summed up; moreover the productivity of genetic engineering strain has been verified. Through the above research, the following results was obtained that the titer of genetic engineering strain WB/3446 and the WB/3* 7301 flask is respectively 973 mg/L and 862 mg/L, and additionally the original strains WB is increased by 39.8% and 33.6% respectively. Through small fermentation experiment, the condition of small fermentation process of 5 L fermentor, which includes temperature being 30℃, the pH value being 7.0-7.2, two-layer stirring paddle, stirring speed being 350 rpm, inoculum concentration being 20%, seed age being about 50 h, the average feeding rate of Glucose being 0.0076 g/min, was respectively determined. It can be summaried, from response surface optimized experiment, that under the condition of the tempurature being 31℃,inculation quantity being 19.5% and the seed age being 49.5h,the fermentation level of WB strain is 3210mg/L,and additionally,three repeated experiments also verify the accuacy of the conclusion. By doing small fermentation experiments of genetically engineered strain with the above-mentioned small fermentation process, the conclusion was reached that the production of erythromycin A of WB/3446 is 4383mg/L, with respect to original strain WB, increased by 35.1%; what’s more, the mycelium of genetic engineering strain WB/3446 grows slightly faster than WB strain, its sugar consuming quantity being larger; and besides the average accelerating rate of glucose is 0.0092g/min, while that of WB strain is 0.0076g/min. The small fermentation results have been concluded, from genetic engineering strain WB/3* 7301, that the production of Erythromycin A is 4177mg/L, increased by 34.6% compared with WB strain; and in addition, there is no any significant differences in its mycelium amount of the pre-fermentation compared with that of WB strain, while, with regard to its mycelium amount of the fermentation in middle and later periods, genetic engineering bacteria WB/3* 7301 bacteria concentration is always higher than the WB strains and the average glucose adding rate is 0.0095 g/min, but there are slightly differences in the adding rate of glucose during various periods. The above results play an important guiding role in the further development of the fermentation and genetic engineering strains amplified experiment research.