利用基因工程定向阻断尼莫克汀工业生产菌株中多个杂质组份的生物合成
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摘要
目的通过基因工程定向改造尼莫克汀(nemadectin)产生菌蓝灰链霉菌(Streptomyces cyaneogriseus subsp.noncyanogenus),以阻断除主要杂质组分合成,提高有效组分产量。方法通过构建和筛选蓝灰链霉菌的基因组文库,获得了与LL-F28249ω合成相关的基因nolB。在蓝灰链霉菌的工业生产菌株NS1-8中连续删除尼莫克汀合成途径的nemD基因和类似寡霉素合成途径的nolB基因,获得了突变株NM-10。结果发酵检测产物表明,NM-10突变株产生活性组分LL-F28249α产量(1312mg/L)高于出发工业菌株NS1-8(1030mg/L),且不再产生LL-F28249γ、LL-F28249λ和LL-F28249ω等3个结构类似的杂质组份。结论 nemD和nolB基因连续敲除可用于改造产尼莫克汀蓝灰链霉菌工业产生菌,降低非有效组分含量并提高产量。
Objective To construct a nemadectin industrial strain with less byproducts by genetically engineering the Streptomyces cyaneogriseus subsp.noncyanogenus.Methods The nolB involved in LL-F28249ωbiosynthesis was cloned through screening the genomic library of Streptomyces cyaneogriseus subsp.noncyanogenus.Mutant strain NM-10 was obtained by sequential deletion of nolB and nemD involved in nemadectin biosynthsis from strain NS1-8.Results The nemadectin production of strain NM-10(1~312mg/L) was higher than that of strain NS1-8(1030mg/L).Furthermore there were no byproducts like LL-F28249γ,LL-F28249λ and LL-F28249ωcould be detected in strain NM-10.Conclusion Sequential deletion of nemD and nolB can be employed to improve production of nemadectin and decrease byproducts in S.cyaneogriseus subsp.noncyanogenus industrial strain.
Objective To construct a nemadectin industrial strain with less byproducts by genetically engineering the Streptomyces cyaneogriseus subsp.noncyanogenus.Methods The nolB involved in LL-F28249ωbiosynthesis was cloned through screening the genomic library of Streptomyces cyaneogriseus subsp.noncyanogenus.Mutant strain NM-10 was obtained by sequential deletion of nolB and nemD involved in nemadectin biosynthsis from strain NS1-8.Results The nemadectin production of strain NM-10(1~312mg/L) was higher than that of strain NS1-8(1030mg/L).Furthermore there were no byproducts like LL-F28249γ,LL-F28249λ and LL-F28249ωcould be detected in strain NM-10.Conclusion Sequential deletion of nemD and nolB can be employed to improve production of nemadectin and decrease byproducts in S.cyaneogriseus subsp.noncyanogenus industrial strain.
引文
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[4]Gust B,Challis G L,Fowler K,et al.PCR-targeted Streptomyces gene replacement identifi es a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin[J].Proc Natl Acad Sci U S A,2003,100(4):1541-1546.
[5]Kieser T,Bibb M J,Buttner M,et al.Practical Streptomyces genetics[M].Norwich:The John Innes Foundation,2000.
[6]夏海洋,黄隽,胡敏杰,等.构建有序排列的除虫链霉菌基因组的柯斯文库用于工业生产菌株的遗传改造[J].中国抗生素杂志,2009,34(7):340-343.
[7]Sambrook J,Fritsch E F,Maniatis T.Molecular cloning:A laboratory manual[M].New York:Cold Spring Harbor Laboratory Press,1989.
[8]Lu Z,Xie P,Qin Z.Promotion of markerless deletion of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor[J].Acta Biochimica et Biophysica Sinica,2010,42(10):717-721.
[9]Omura S,Ikeda H,Ishikawa J,et al.Genome sequence of an industrial microorganism Streptomyces avermitilis:deducing the ability of producing secondary metabolites[J].Proc Natl Acad Sci U S A,2001,98(21):12215-12220.
[10]Sun Y,Zhou X,Liu J,et al.'Streptomyces nanchangensis',a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin,contains multiple polyketide gene clusters[J].Microbiology,2002,148(2):361-371.
[11]Tsou H R,Ahmed Z H,Fiala R R,et al.Biosynthetic origin of the carbon skeleton and oxygen atoms of the LL-F28249alpha,a potent antiparasitic macrolide[J].J Antibiot,1989,42(3):398-406.
[2]Asato G,France D.23-Imino derivatives of LL-F28249compounds:US,1990/4916154[P].1990-04-10.
[3]Huang C J,Chaleff D T,Ruppen M E,et al.Cloning genes from Streptomyces cyaneogriseus subsp.noncyanogenus for biosynthesis of antibiotics and methods of use:US,2008/7396660[P].2008-07-08.
[4]Gust B,Challis G L,Fowler K,et al.PCR-targeted Streptomyces gene replacement identifi es a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin[J].Proc Natl Acad Sci U S A,2003,100(4):1541-1546.
[5]Kieser T,Bibb M J,Buttner M,et al.Practical Streptomyces genetics[M].Norwich:The John Innes Foundation,2000.
[6]夏海洋,黄隽,胡敏杰,等.构建有序排列的除虫链霉菌基因组的柯斯文库用于工业生产菌株的遗传改造[J].中国抗生素杂志,2009,34(7):340-343.
[7]Sambrook J,Fritsch E F,Maniatis T.Molecular cloning:A laboratory manual[M].New York:Cold Spring Harbor Laboratory Press,1989.
[8]Lu Z,Xie P,Qin Z.Promotion of markerless deletion of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor[J].Acta Biochimica et Biophysica Sinica,2010,42(10):717-721.
[9]Omura S,Ikeda H,Ishikawa J,et al.Genome sequence of an industrial microorganism Streptomyces avermitilis:deducing the ability of producing secondary metabolites[J].Proc Natl Acad Sci U S A,2001,98(21):12215-12220.
[10]Sun Y,Zhou X,Liu J,et al.'Streptomyces nanchangensis',a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin,contains multiple polyketide gene clusters[J].Microbiology,2002,148(2):361-371.
[11]Tsou H R,Ahmed Z H,Fiala R R,et al.Biosynthetic origin of the carbon skeleton and oxygen atoms of the LL-F28249alpha,a potent antiparasitic macrolide[J].J Antibiot,1989,42(3):398-406.