CAJ | 학술논문

目的：在红色糖多孢菌（ S．erythraea，SE）中串联表达S-腺苷甲硫氨酸合成酶基因metK、透明颤菌血红蛋白基因vhbS和多效调控蛋白基因adpA，构建红霉素高产的工程菌株。方法通过PEG介导的原生质体转化方法，将携带metK、vhbS和adpA基因片段的整合型质粒导入红霉素野生菌株SE的A226及工业菌株WB中，安普霉素抗性筛选与PCR鉴定获得工程菌株，并利用枯草芽孢杆菌抑菌实验与高效液相色谱分析比较出发菌株及其工程菌株的红霉素产量。结果与结论成功构建4株A226衍生菌株A226-P1～P4与3株WB衍生菌株WB-P1～P3。相比野生菌株A226，工程菌株A226-P1～P4的相对红霉素效价提高了8％～25％，其红霉素A产量增加了64％～94％。同样，工程菌株WB-P1～P3的相对红霉素效价与红霉素A产量较出发菌株WB都有明显提高，分别增加了6％～10％与31％～62％。说明metK、vhbS和adpA的串联表达提高SE的红霉素产量具有普适性。
목적：재홍색당다포균（ S．erythraea，SE）중천련표체S-선감갑류안산합성매기인metK、투명전균혈홍단백기인vhbS화다효조공단백기인adpA，구건홍매소고산적공정균주。방법통과PEG개도적원생질체전화방법，장휴대metK、vhbS화adpA기인편단적정합형질립도입홍매소야생균주SE적A226급공업균주WB중，안보매소항성사선여PCR감정획득공정균주，병이용고초아포간균억균실험여고효액상색보분석비교출발균주급기공정균주적홍매소산량。결과여결론성공구건4주A226연생균주A226-P1～P4여3주WB연생균주WB-P1～P3。상비야생균주A226，공정균주A226-P1～P4적상대홍매소효개제고료8％～25％，기홍매소A산량증가료64％～94％。동양，공정균주WB-P1～P3적상대홍매소효개여홍매소A산량교출발균주WB도유명현제고，분별증가료6％～10％여31％～62％。설명metK、vhbS화adpA적천련표체제고SE적홍매소산량구유보괄성。
Objective To construct erythromycin-overproducing mutants by tandemly expressing S-adenosylmethionine synthetase gene metK, Vitreoscilla hemoglobin gene vhbS and pleiotropic regulatory gene adpA in Saccharopolyspora eryth-raea.Methods Through PEG-mediated protoplast transformation , the integrative plasmid carrying metK, vhbS and adpA was respectively introduced into erythromycin-producing wild-type strain S.erythraea A226 and industrial strain WB .The engineered strains were generated by apramycin resistance screening and PCR identification .The erythromycin production was compared in original strains and their mutants by the inhibition test of Bacillus subtilis and HPLC analysis .Results and Conclusion Four A226-derived mutants A226-P1-P4 and three WB-derived mutants WB-P1-P3 were independently obtained.Compared with wild-type strain A226, the relative erythromycin titer of the four engineered strains A 226-P1-P4 was increased from 8%to 25%by scoring the growth-inhibition zones .Further HPLC analysis showed that the four mutants had increased erythromycin A yield by 64%-94%.Likewise, the relative erythromycin titer and erythromycin A yield of the three engineered strains WB-P1-P3 were enhanced by 6%-10%and 31%-62%, respectively, in comparison with the original strain WB.The results show the universality of enhancing erythromycin productionvia tandem expression of metK, vhbS and adpA in S.erythraea.