核糖体工程技术选育米尔贝霉素高产菌株
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摘要
本实验室中保存的一株从土壤中分离得到的米尔贝链霉菌(Streptomyces milbemycinicus)27号菌株所产米尔贝霉素A3和A4的初始产量分别为61.0和23.8μg/m L。以该菌株为出发菌株,采用核糖体工程技术,结合紫外诱变技术,对米尔贝霉素产生菌米尔贝链霉菌进行诱变,并以链霉素耐受为筛选压力进行筛选。经过诱变后对单菌落进行摇瓶复筛,其中正突变株中米尔贝霉素产量最高的菌株编号是R2-6-5,其米尔贝霉素A3和A4的产量分别是105.2和38.8μg/m L,较原始菌株分别提高了72.5%和63.3%,且遗传稳定。最后,对产量变化较大的11株突变株基因组中rsm G基因和rsp L基因进行突变位点分析,发现在rsp L基因内均未发生突变,rsm G基因均发生突变。本研究表明,链霉素抗性降低的突变菌株确实都在相关基因内发生突变,且核糖体工程结合紫外诱变的诱变方式效果良好,能够快速有效的提高米尔贝链霉菌生物合成米尔贝霉素的能力。
A Streptomyces milbemycinicus strain isolated from the soil with an initial yield of milbemycin A3 and A4 were obtained by natural selection. The yields were 61.0 and 23.8μg/m L, respectively. The strain was used as a starting strain, the ribosomal engineering technique and the UV mutagenesis technique were used to mutate the milbemycin-producing strain. Streptomycin was used as the screening pressure. Forty-two mutants were obtained. Among them, R2-6-5 had a high yield of milbemycins, and the yield of milbemycin A3 and A4 was 105.2 and 38.8μg/m L, respectively. The yields were 72.5% and 63.3% higher than those of the original strain. Finally, rsp L and rsm G gene were detected in the genome of 11 mutants which had a big impact on the yields. The rsm G gene was found to be mutated in all detected strains and there is no change in rsp L gene. The combination mutation method of ribosomal engineering and UV, with streptomycin as the selection factor for breeding had a high positive mutation rate, and the yield of milbemycins was significantly increased. This study indicated that mutant strains with reduced streptomycin resistance all had mutated in the relevant genes, and the mutagenesis of ribosomal engineering combined with UV mutagenesis was effective and could improve the biosynthesis of milbemycins.
A Streptomyces milbemycinicus strain isolated from the soil with an initial yield of milbemycin A3 and A4 were obtained by natural selection. The yields were 61.0 and 23.8μg/m L, respectively. The strain was used as a starting strain, the ribosomal engineering technique and the UV mutagenesis technique were used to mutate the milbemycin-producing strain. Streptomycin was used as the screening pressure. Forty-two mutants were obtained. Among them, R2-6-5 had a high yield of milbemycins, and the yield of milbemycin A3 and A4 was 105.2 and 38.8μg/m L, respectively. The yields were 72.5% and 63.3% higher than those of the original strain. Finally, rsp L and rsm G gene were detected in the genome of 11 mutants which had a big impact on the yields. The rsm G gene was found to be mutated in all detected strains and there is no change in rsp L gene. The combination mutation method of ribosomal engineering and UV, with streptomycin as the selection factor for breeding had a high positive mutation rate, and the yield of milbemycins was significantly increased. This study indicated that mutant strains with reduced streptomycin resistance all had mutated in the relevant genes, and the mutagenesis of ribosomal engineering combined with UV mutagenesis was effective and could improve the biosynthesis of milbemycins.
引文
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[7]海乐,黄宇琪,廖国建,等.放线菌Streptomyces sp.FJ3的核糖体工程改良与活性产物的分离[J].微生物学报,2011,51(7):934-940.
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[9]韩晓,崔承彬,韩小贤,等.放线菌野生株代谢功能的核糖体工程改造与新产抗肿瘤活性产物研究[J].国际药学研究杂志,2009,36(6):435-442.
[10]李文华,黄永春,杨少彬,等.核糖体工程技术选育卡伍尔链霉菌高产菌株[J].中国农学通报,2017,33(7):48-53.
[11]李晓玲,游中元,徐俊,等.红树林来源白浅灰链霉菌中生物碱类次级代谢产物研究及核糖体工程优化[J].台湾海峡,2012,31(1):143-149.
[12]吴光耀,陈旭升,王靓,等.核糖体工程技术选育ε-聚赖氨酸高产菌株[J].微生物学通报,2016,43(12):2744-2751.
[13]Okamoto-Hosoya Y,Okamoto S,Ochi K.Development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsL gene in Streptomyces lividans[J].Appl Environ Microbiol,2003,69(7):4256-4259.
[14]Okamoto S,Tamaru A,Nakajima C,et al.Loss of a conserved 7-methylguanosine modification in 16S rRNAconfers low-level streptomycin resistance in bacteria[J].Mol Microbiol,2007,63(4):1096-1106.
[15]Nishimura K,Hosaka T,Tokuyama S,et al.Mutations in rsmG,encoding a 16S rRNA methyltransferase,result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)[J].JBacteriol,2007,189(10):3876-3883.
[16]Takiguchi Y,Mishima H,Okuda M,et al.Milbemycins,a new family of macrolide antibiotics:fermentation,isolation and physico-chemical properties[J].J Antiobiot,1980,33(10):1120-1127.
[17]Zhang J,An J,Wang J J,et al.Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin[J].Appl Microbiol Biotechnol,2013,97(23):10091-10101.
[18]Wang X J,Zhang B,Yan Y J,et al.Characterization and analysis of an industrial strain of Streptomyces bingchenggensis by genome sequencing and gene microarray[J].Genome,2013,56(11):677-689.
[2]Kumar Y,Goodfellow M.Reclassification of Streptomyces hygroscopicus strains as Streptomyces aldersoniae sp.nov.,Streptomyces angustmyceticus sp.nov.,comb.nov.,Streptomyces ascomycinicus sp.nov.,Streptomyces decoyicus sp.nov.,comb.nov.,Streptomyces milbemycinicus sp.nov.and Streptomyces wellingtoniae sp.nov[J].Int J Sys Evol Microbiol,2010,60(4):769-775.
[3]Xiang W,Wang J,Wang X,et al.Further new milbemycin antibiotics from Streptomyces bingchenggensis[J].JAntibiot,2007,60(10):608-613.
[4]Baltz R H.Genetic manipulation of antibioticproducing Streptomyces[J].Trends Microbiol,1998,6(2):76-83.
[5]陈丽杰,商光来,袁文杰,等.利用核糖体工程选育丙酮丁醇菌提高丁醇产量[J].生物工程学报,2012,28(9):1048-1058.
[6]陈丽仙.核糖体工程技术在刺糖多孢菌上的初步应用[D].福州:福建农林大学,2010.
[7]海乐,黄宇琪,廖国建,等.放线菌Streptomyces sp.FJ3的核糖体工程改良与活性产物的分离[J].微生物学报,2011,51(7):934-940.
[8]韩小贤,崔承彬,姚志伟,等.陆地与海洋来源放线菌次级代谢能力的核糖体工程改造[J].中国海洋大学学报(自然科学版),2010,40(5):47-52.
[9]韩晓,崔承彬,韩小贤,等.放线菌野生株代谢功能的核糖体工程改造与新产抗肿瘤活性产物研究[J].国际药学研究杂志,2009,36(6):435-442.
[10]李文华,黄永春,杨少彬,等.核糖体工程技术选育卡伍尔链霉菌高产菌株[J].中国农学通报,2017,33(7):48-53.
[11]李晓玲,游中元,徐俊,等.红树林来源白浅灰链霉菌中生物碱类次级代谢产物研究及核糖体工程优化[J].台湾海峡,2012,31(1):143-149.
[12]吴光耀,陈旭升,王靓,等.核糖体工程技术选育ε-聚赖氨酸高产菌株[J].微生物学通报,2016,43(12):2744-2751.
[13]Okamoto-Hosoya Y,Okamoto S,Ochi K.Development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsL gene in Streptomyces lividans[J].Appl Environ Microbiol,2003,69(7):4256-4259.
[14]Okamoto S,Tamaru A,Nakajima C,et al.Loss of a conserved 7-methylguanosine modification in 16S rRNAconfers low-level streptomycin resistance in bacteria[J].Mol Microbiol,2007,63(4):1096-1106.
[15]Nishimura K,Hosaka T,Tokuyama S,et al.Mutations in rsmG,encoding a 16S rRNA methyltransferase,result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)[J].JBacteriol,2007,189(10):3876-3883.
[16]Takiguchi Y,Mishima H,Okuda M,et al.Milbemycins,a new family of macrolide antibiotics:fermentation,isolation and physico-chemical properties[J].J Antiobiot,1980,33(10):1120-1127.
[17]Zhang J,An J,Wang J J,et al.Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin[J].Appl Microbiol Biotechnol,2013,97(23):10091-10101.
[18]Wang X J,Zhang B,Yan Y J,et al.Characterization and analysis of an industrial strain of Streptomyces bingchenggensis by genome sequencing and gene microarray[J].Genome,2013,56(11):677-689.