低氮源消耗和高产雷帕霉素菌株的选育
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摘要
目的采用亚硝基胍(NTG)、常压室温等离子体(ARTP)、核糖体工程育种方法处理产雷帕霉素游动放线菌SIIA-1602,结合OSMAC策略以期筛选得到发酵水平有较大提高,氮源利用更加高效的新菌株。方法首轮采用NTG诱变筛选;第二轮采用链霉素抗性育种,第三轮采用ARTP诱变育种;采用3种不同的发酵培养基考察突变菌株的发酵水平。结果出发株游动放线菌SIIA-1602经过NTG诱变、链霉素抗性和ARTP诱变筛选得到的突变株ASN-256,其发酵水平提高了55.7%,通过发酵培养基删减有机复合氮源并且添加了赖氨酸和哌可酸后,发酵水平提高分别107.6%和148.9%。在10t发酵罐通过流加,使菌种ASN-256发酵单位进一步提高到1250mg/L,是出发菌株SIIA-1602发酵水平的2.85倍。菌种ASN-256发酵过程中总氮源使用量减少50%,放罐时氨基氮排放仅为菌株SIIA-1602的一半,菌渣量也显著降低。结论本方法简单经济,突变效率高,能够快速获得传代稳定,氮源利用更加高效的雷帕霉素高产突变株。另外,该菌通过发酵培养基的调整实现了氨基氮排放和菌渣量的大幅降低,在环保方面取得了显著的效益。
Objective To improve the production of rapamycin and the utilization rate of nitrogen source, actinoplanes SIIA-1602 strain was treated with NTG(1-methyl-3-nitro-1-nitroso-guanidin), ARTP(atmospheric and room temperature plasma) or ribosome engineering breeding by OSMAC strategies. Methods Actinoplanes SIIA-1602 strain was treated with NTG at the first screening. Then, the mutant strains were treated with streptomycinresistance breeding at the second screening. Subsequently, the mutant strains were treated with ARTP to improve breeding efficiency at the third screening. The optimal fermentation level of mutated strains was selected from three different fermentation mediums. Results The mutant stain ASN-256 was obtained by NTG, ARTP and streptomycinresistance screening. Its potency of rapamycin production was increased by 55.7% compared with SIIA-1602 strain. In addition, the fermentation level of ASN-256 strains was increased by 107.6% and 148.9% through decreasing the complex nitrogen source and adding lysine and pipecolic acid. Furthermore, the fermentation titer was 1250 mg/L at 10 tons-fermenter with fed batch of pipecolic acid. The fermentation level of ASN-256 strain was 2.85 folds higher than that of SIIA-1602 strain. The total nitrogen utilization and amino nitrogen emission of ASN-256 strains were decreased by 50% compared with that of SIIA-1602 strain. The bacterial residue amount of ASN-256 strain was also significantly decreased compared with that of SIIA-1602 strain. Conclusion This screening method was simple and low-cost, which could rapidly obtain the mutant strain with high production of rapamycin, the high genetic stability and high utilization rate of nitrogen. In addition, the reduction of nitrogen emission has obtained significant environmental benefits.
Objective To improve the production of rapamycin and the utilization rate of nitrogen source, actinoplanes SIIA-1602 strain was treated with NTG(1-methyl-3-nitro-1-nitroso-guanidin), ARTP(atmospheric and room temperature plasma) or ribosome engineering breeding by OSMAC strategies. Methods Actinoplanes SIIA-1602 strain was treated with NTG at the first screening. Then, the mutant strains were treated with streptomycinresistance breeding at the second screening. Subsequently, the mutant strains were treated with ARTP to improve breeding efficiency at the third screening. The optimal fermentation level of mutated strains was selected from three different fermentation mediums. Results The mutant stain ASN-256 was obtained by NTG, ARTP and streptomycinresistance screening. Its potency of rapamycin production was increased by 55.7% compared with SIIA-1602 strain. In addition, the fermentation level of ASN-256 strains was increased by 107.6% and 148.9% through decreasing the complex nitrogen source and adding lysine and pipecolic acid. Furthermore, the fermentation titer was 1250 mg/L at 10 tons-fermenter with fed batch of pipecolic acid. The fermentation level of ASN-256 strain was 2.85 folds higher than that of SIIA-1602 strain. The total nitrogen utilization and amino nitrogen emission of ASN-256 strains were decreased by 50% compared with that of SIIA-1602 strain. The bacterial residue amount of ASN-256 strain was also significantly decreased compared with that of SIIA-1602 strain. Conclusion This screening method was simple and low-cost, which could rapidly obtain the mutant strain with high production of rapamycin, the high genetic stability and high utilization rate of nitrogen. In addition, the reduction of nitrogen emission has obtained significant environmental benefits.
引文
[1]杨冬.雷帕霉素高产菌株的选育[D].杭州:浙江大学,2006.
[2]Balter M.Protein matchmaker may lead new gene therapy to the altar[J].Science,1996,273(5272):183.
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[4]Ochi K.From microbial differentiation to ribosome engineering[J].Biosci Biotechnol Biochem,2007,71(6):1373-1386.
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[6]王耀耀,刘云清,朱研研,等.组合链霉素和利福平抗性突变去甲基万古霉素高产菌株的选育杂志[J].中国抗生素杂志,2006,31(4):243-246.
[7]刘俊慧.基于OSMAC策略的海洋微生物次级代谢产物研究[D].大连:大连理工大学,2005.
[8]高芳霞,田敏,雷叶明,等.微生物来源的缩肽类化合物enopeptin A的研究(I):菌种选育及发酵工艺研究[J].中国抗生素杂志,2018,43(4):429-436.
[9]石漫漫,乔长晟,朱明,等.高产雷帕霉素的游动放线菌菌种的选育[J].中国生物工程杂志,2015,35(2):72-77
[10]王耀耀.雷帕霉素产生菌的诱变育种及发酵条件的优化[D].石家庄:河北医科大学,2009.
[11]杨国新,陈夏琴,余辉,等.雷帕霉素二醛的分离和结构鉴定[J].中国抗生素杂志,2018,43(4):450-456.
[2]Balter M.Protein matchmaker may lead new gene therapy to the altar[J].Science,1996,273(5272):183.
[3]Hidalgo M,Rowinsky E K.The rapamycin-sensitive signal transduction pathway as a target for cancer therapy[J].Oncogene,2000,19(56):6680-6686.
[4]Ochi K.From microbial differentiation to ribosome engineering[J].Biosci Biotechnol Biochem,2007,71(6):1373-1386.
[5]谢庶洁,肖静,徐俊.微生物核糖体工程研究进展[J].微生物学报,2009,49(8):981-986.
[6]王耀耀,刘云清,朱研研,等.组合链霉素和利福平抗性突变去甲基万古霉素高产菌株的选育杂志[J].中国抗生素杂志,2006,31(4):243-246.
[7]刘俊慧.基于OSMAC策略的海洋微生物次级代谢产物研究[D].大连:大连理工大学,2005.
[8]高芳霞,田敏,雷叶明,等.微生物来源的缩肽类化合物enopeptin A的研究(I):菌种选育及发酵工艺研究[J].中国抗生素杂志,2018,43(4):429-436.
[9]石漫漫,乔长晟,朱明,等.高产雷帕霉素的游动放线菌菌种的选育[J].中国生物工程杂志,2015,35(2):72-77
[10]王耀耀.雷帕霉素产生菌的诱变育种及发酵条件的优化[D].石家庄:河北医科大学,2009.
[11]杨国新,陈夏琴,余辉,等.雷帕霉素二醛的分离和结构鉴定[J].中国抗生素杂志,2018,43(4):450-456.
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