离子注入选育霉酚酸高产菌株及其发酵条件研究

离子注入技术是20世纪80年代兴起的一种综合诱变技术,其应用于生物工程已取得了丰硕成果,但在霉酚酸产生菌的诱变育种中的应用还未见报道。短密青霉菌(Penicillium brevicompactum)M_51是从土壤中分离得到的MPA产生菌F_663经过紫外线、微波等诱变处理得到的。为获得霉酚酸的高产工业菌株,进一步对该菌株进行了离子注入诱变处理。用15keV氮离子分5个剂量进行处理,结果显示,随离子注入剂量增加,存活率呈现较明显的下降_上升_下降的“马鞍型”变化趋势。在剂量为140×2.6×1013ions/cm2时,菌株变异率及正变率均最高,分别达到88.9%和63.4%。用HPLC定量测定发酵液中霉酚酸的含量,筛选到产霉酚酸能力提高30.1%的突变株M_163。经过连续传代试验,其遗传性状稳定。对发酵条件的优化结果显示最佳种龄为24h;用正交试验方法对发酵培养基中的碳、氮源进行优化,得到较优配方。突变株M_163在最优发酵条件下,霉酚酸摇瓶发酵单位可达2819μg/mL。野生菌株F_663的MPA产量为133μg/mL,经过5代诱变育种及发酵条件优化,产量提高了20.2倍。

Studies on the breeding by ion implantation and cultivation of mycophenolic acid producing strain

Mycophenolic acid is produced by aerobic fermentation of several Penicillium species. It has a broad spectrum of activity like antitumor activity, antiviral, anti-psoriatic, immunosuppressive and anti-inflammatory activity. It also exhibits antibacterial and antifungal activities. The immunosuppressive effect of MPA has been important in treatment of organ rejection after organ transplant surgery. There is a continuous need to find improved process for efficiently obtaining superior MPA producing mutants. In recent years, the ion implantation technique has been widely applied in many fields and has been drawn morn concern. However there is no report in the field of mutational breeding of MPA producing strain. Penicillium brevicompactum M-51 was derived from MPA producing strain F-663 by varied mutational methods including U.V. and microwave irradiation. In the process of increasing the production of MPA from P. brevicompactum M-51, a mutant strain M-163 was obtained by means of N+ ion implantation. An decline-increase-decline tendency of strain survival rates was observed when the strain was implanted by N+ ion with dose from 20 2.6 x 10(13) ions/cm2 to 180 x 2.6 x 10(13) ions/cm2 under implantation energy 15 keV. It apparently appeared "saddle shape". And under the implantation dose of 140 x 2.6 x 10(13) ions/cm2, the variation rate and the positive variation rate of the strain had reached the highest values 88.9% and 63.4%, respectively. The HPLC results showed that MPA yield of P. brevicompactum M-163 was improved by 30.1%, and its productivity was rather stable through successive transfer of cultures. The effect of seed growth time on yield of MPA was studied, and the best seed age was 24h after incubation. In the mean time, the fermentative condition of M-163 was studied through orthogonal design. The major ingredients being investigated included carbon and nitrogen sources. Finally the optimized fermentation medium was obtained. The yield of MPA reached 2819g/mL in the optimized submerged fermentation progress. In conclusion, N+ ion implantation had been proven to be effective for mutational breeding of P. brevicompactum M-51. By means of N+ ion implantation and optimizing the fermentative condition, the yield of MPA was increased by 20.2 times than the wild-type stain F-663.