pheS , an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens.
pheS , an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens.
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pheS ，一种有效的独立于宿主基因型的反选择标记，用于解淀粉芽孢杆菌中无标记染色体删除。
DOI：
10.1007/s00253-016-7906-9
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Yan Xin
中科院分区：
文献类型：
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作者：
Zhou Chaoyang;Shi Lingling;Ye Bin;Feng Haichao;Zhang Ji;Zhang Ruifu;Yan Xin
除用于生产商业酶和代谢物外，解淀粉芽孢杆菌也是一组重要的促进植物生长的根瘤菌，支持植物生长并抑制植物病原体。一个不依赖于宿主基因型的反选择标记可以实现快速的基因操作和代谢工程，从而加快b型疟原虫的研究。解淀粉酶及其作为微生物细胞工厂和促进植物生长的根瘤菌的发展。本文通过对枯草芽孢杆菌菌株168中苯丙烯酰trna合成酶α-亚基的基因进行点突变，构建了一个与宿主基因型无关的反选择标记es *。在5mmp -氯苯丙氨酸存在下，100%的b。解淀粉酶菌株SQR9携带phes *的细胞被杀死，而野生型菌株SQR9表现出对上氯苯丙氨酸的抗性。我们采用了一种基于simpleses *和重叠pcr的策略来创建amyegene的无标记缺失以及b中的37-kbbmycluster。amyloliquefaciensSQR9。phes *作为b型抗选择标记物的有效性。通过在菌株sb中缺失淀粉基因进一步证实了其解淀粉酶活性。解淀粉剂fzb42和NJN-6。此外，还初步评估了phes *在其他芽孢杆菌中的潜在应用。PheS*在b中的表达。枯草杆菌168和b。该菌株对两种宿主的顶氯苯丙氨酸均有明显的敏感性，表明phes *可作为这两种菌株的反选择标记物（CSM）。
Aside from applications in the production of commercial enzymes and metabolites,Bacillus amyloliquefaciensis also an important group of plant growth-promoting rhizobacteria that supports plant growth and suppresses phytopathogens. A host-genotype-independent counter-selectable marker would enable rapid genetic manipulation and metabolic engineering, accelerating the study ofB. amyloliquefaciensand its development as both a microbial cell factory and plant growth-promoting rhizobacteria. Here, a host-genotype-independent counter-selectable markerpheS*was constructed through a point mutation of the genepheS, which encodes theα-subunit of phenylalanyl-tRNA synthetase inBacillus subtilisstrain 168. In the presence of 5 mMp-chloro-phenylalanine, 100 % ofB. amyloliquefaciensstrain SQR9 cells carryingpheS*were killed, whereas the wild-type strain SQR9 showed resistance top-chloro-phenylalanine. A simplepheS*and overlap-PCR-based strategy was developed to create the marker-free deletion of theamyEgene as well as a 37-kbbmycluster inB. amyloliquefaciensSQR9. The effectiveness ofpheS*as a counter-selectable marker inB. amyloliquefacienswas further confirmed through the deletion ofamyEgenes in strainsB. amyloliquefaciensFZB42 and NJN-6. In addition, the potential use ofpheS*in otherBacillusspecies was preliminarily assessed. The expression of PheS*inB. subtilisstrain 168 andB. cereusstrain ATCC 14579 caused pronounced sensitivity of both hosts top-chloro-phenylalanine, indicating thatpheS*could be used as a counter-selectable marker (CSM) in these strains.