CN107267434B - 一种高产达托霉素的玫瑰孢链霉菌l31及其构建方法 - Google Patents
一种高产达托霉素的玫瑰孢链霉菌l31及其构建方法 Download PDFInfo
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Abstract
本发明涉及一种高产达托霉素的玫瑰孢链霉菌(Streptomyces roseosporus)L31，它是通过融合达托霉素合成途径中起始反应的两个酶(DptE和DptF)，并定向改造色氨酸代谢途径获得的，该菌株达托霉素产量比出发菌株(WT)提高了140％。
Description
技术领域
本发明涉及微生物制药领域，特别涉及一种高产达托霉素的玫瑰孢链霉菌L31及其构建方法。
背景技术
由于抗生素在临床上的广泛使用，病原菌的耐药性也变得越来越严重，人类迫切需要新型的抗生素来对抗耐药菌的产生。达托霉素是一种从玫瑰孢链霉菌(Streptomycesroseosporus)发酵液中提取的环脂肽类抗生素，对革兰氏阳性菌有很强的抗菌活性，被认为是万古霉素的最佳替代品。达托霉素与病原菌细胞膜结合，使离子外流导致其死亡。因其独特的环脂肽结构和作用机制，以及不与其它抗生素产生交叉耐药性，达托霉素具有广泛的应用前景。
2003年9月，达托霉素的注射剂Cubicin，作为全新结构的抗生素被美国FDA批准上市，是第一种被批准应用于临床的脂肽类抗生素，临床上达托霉素用于治疗由耐万古霉素肠球菌(VRE)，耐甲氧西林金黄色葡萄球菌(MRSA)，凝固酶阴性葡萄球菌(CNS)，耐青霉素肺炎链球菌(PRSP)等引起的复杂性皮肤病和皮肤结构感染。2014年全球该抗生素总销售超12亿美元，2015年我国有四家药企获CFDA的新药证书。
但目前达托霉素的发酵单位低，不能满足工业化生产和临床应用的需求。
发明内容
本发明的目的在于针对现有技术的不足，提供一种高产达托霉素的玫瑰孢链霉菌L31及其构建方法。
本发明的目的是通过以下技术方案实现的：(1)用SEQ ID NO.1所述的核苷酸序列，对出发菌株WT基因组中与SEQ ID NO.1同源的序列进行替换，获得中间菌株1。(2)用SEQID NO.2所述的核苷酸序列，对中间菌株1基因组中与SEQ ID NO.2同源的序列进行替换，获得中间菌株2。(3)将SEQ ID NO.3所述的核苷酸序列，整合到中间菌株2基因组的attB位点，获得最终的菌株玫瑰孢链霉菌L31。
本发明所提供的玫瑰孢链霉菌L31，已于2017年6月12日保藏于中国微生物菌种保藏委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号，保藏登记号为：CGMCC No.14233，分类名为：玫瑰孢链霉菌(Streptomyces roseosporus)。
本发明的有益效果在于：本发明通过定向改造色氨酸代谢途径，提高犬尿氨酸的供应，并融合达托霉素合成途径中起始反应的两个酶(DptE和DptF)，加快起始反应的速率，从而提高达托霉素的产量。
附图说明
图1为L31和WT菌株发酵产量对比。
具体实施方式
本发明通过基因工程手段，在基因组中融合dptE和dptF的编码区，从而融合DptE和DptF获得酶复合体，并鉴定出了玫瑰孢链霉菌中色氨酸代谢相关的基因，通过基因工程手段，敲除犬尿氨酸的降解的基因，高表达犬尿氨酸生成的基因，获得了达托霉素产量提高的基因工程菌株玫瑰孢链霉菌L31。
该菌株连续多次在斜面培养基传代，经种子培养基和发酵培养基进行发酵实验验证，通过高效液相色谱(HPLC)测定发酵液中达托霉素的发酵单位，结果显示玫瑰孢链霉菌L31的遗传性状稳定，达托霉素产量提高了140％。
下面结合实施例对本发明作进一步说明。
实施例1
本实施例中所使用的培养基：
1、R5固体培养基：蔗糖10.3％，葡萄糖1％，酵母提取物0.5％，酪蛋白水解物0.01％，K2SO4 0.025％，MgCl2·6H2O 1.012％，微量元素溶液0.2％，三羟甲基甲胺基乙磺酸0.573％，脯氨酸0.03％，琼脂粉2.2％。灭菌后，每1L培养基依次加入5％KH2PO4 1mL，5MCaCl2 4mL，10M NaOH 700μL，其余为水，所述百分含量均为质量百分含量，pH自然
微量元素溶液：CuCl2·2H2O 0.1g，Na2B4O7·10H2O 0.1g，MnCl2·4H2O 0.1g，ZnCl20.4g，(NH4)Mo7O24·4H2O 0.1g，FeCl3·6H2O 2g，加水定容至1L
2、TSB培养基：胰蛋白胨大豆肉汤2％，聚乙二醇(PEG600)5％，其余为水，所述百分含量均为质量百分含量，pH自然。
3、YEME发酵培养基：酵母提取物0.3％，蛋白胨0.5％，麦芽提取物0.3％，葡萄糖4％，其余为水，所述百分含量均为质量百分含量，pH自然。
4、MS固体培养基：甘露醇2％，黄豆粉2％，琼脂糖2％，10mMMgCl2其余为水，所述百分含量均为质量百分含量，pH自然。
5、LB液体培养基：NaCl1％，酵母提取物0.5％，蛋白胨1％，其余为水，所述百分含量均为质量百分含量，pH自然
6、2×YT液体培养基：NaCl 0.4％，酵母提取物1％，蛋白胨1.6％，其余为水，所述百分含量均为质量百分含量，pH自然。
步骤如下：
(1)把SEQ ID NO.1所示的DNA序列用聚合酶链式反应(PCR)扩增，扩增后的DNA片段通过DNA限制性内切酶消化后插入大肠杆菌-链霉菌穿梭质粒pKC1139(Bierman,Loganet al.1992)中，再将上述质粒转化到含有质粒pUZ8002大肠杆菌ET12567(Macneil andKlapko 1987)中。
(2)将含有上述质粒的大肠杆菌ET12567(已含有质粒pUZ8002)接种到20ml LB液体培养基(含氯霉素、卡那霉素和阿泊拉霉素)中，37℃，220rpm摇床培养至OD600为0.6，离心收集菌体，用1ml LB液体培养基洗涤四次，用0.5ml的2×YT液体培养基重悬；
(3)将出发菌株(WT)的孢子接入35mL TSB液体培养基中，30℃，250rpm摇床培养36h，离心收集菌体，留体积约50μL的菌体，用1ml 2×YT液体培养基洗涤四次，用0.5ml的2×YT液体培养基重悬；将上述的0.5ml大肠杆菌悬液和0.5ml出发菌株悬液完全混合后均匀涂在MS固体培养基上，30℃培养16小时后，涂布20μg/mL的萘啶酸和25μg/mL的阿泊拉霉素，30℃继续培养4-5天后可长出白色转化子；
(3)将上述得到的链霉菌转化子在含有阿泊拉霉素的R5培养基上培养4-5天后，通过PCR方法确证单交换，将筛选得到的单交换菌株连续传代4-5代以后，筛选阿泊拉霉素敏感的菌株，通过PCR方法确证SEQ ID NO.1的DNA序列已经完全替换出发菌株的基因组中与之同源的序列，获得中间菌株1。
(4)把SEQ ID NO.2所示的DNA序列用聚合酶链式反应(PCR)扩增，通过上述相似的方法，利用SEQ ID NO 2的DNA序列替换到中间菌株1的基因组中与之同源的序列，获得中间菌株2。
(5)把SEQ ID NO.3所示的DNA序列用聚合酶链式反应(PCR)扩增，扩增后的DNA片段通过DNA限制性内切酶消化后插入含有整合酶的大肠杆菌-链霉菌穿梭质粒pIJ8661(Liu,Yuan et al.2015)之中，再将上述质粒转化到大肠杆菌ET12567(已含有质粒pUZ8002)之中。利用上述的方法将穿梭质粒转导至中间菌株2中，在整合酶的作用下SEQ IDNO 3的DNA序列整合到中间菌株2的基因组的attB位点中，获得最终的菌株玫瑰孢链霉菌L31。通过聚合酶链式反应(PCR)扩增，并将扩增产物进行DNA测序，证实该菌株玫瑰孢链霉菌L31包含SEQ ID NO.1～SEQ ID NO.3所述的核苷酸序列。
将出发菌株(WT)和上述步骤5获得的玫瑰孢链霉菌的孢子接种到TSB培养基中，30℃，250rpm摇床培养36h；将1mL的TSB培养基的菌丝体接种至YEME发酵培养基中，30℃，250rpm摇床培养，第36h开始，每12h补加35μL补料液(癸酸:油酸甲酯＝1:1)，在培养48，72，96，120小时后分别取样，发酵液12000rpm离心，上清用millipore滤膜过滤后用于检测达托霉素的产量。
HPLC检测达托霉素的产量的参数如下：
柱子：Zorbax，SB-C18，5μm，4.6mm*250mm，Agilent
仪器：Agilent 1260Infinity
流动相：A相：(0.05M Na2HPO4，pH＝3.15±0.05)
B相：100％乙腈；
A相：B相＝67：35
流速：1.0mL/min
检测波长：222nm
培养48，72，96，120小时的产量如图1所示，从图中可以看出：在培养过程中，基因工程改造菌株玫瑰孢链霉菌L31的达托霉素产量都高于出发菌株(WT)，玫瑰孢链霉菌L31发酵培养在96小时后，达托霉素的产量达到74mg/L，比出发菌株(WT)的产量(26mg/L)提高1.4倍。
SEQUENCE LISTING
<110> 浙江大学
<120> 一种高产达托霉素的玫瑰孢链霉菌L31及其构建方法
<160> 3
<170> PatentIn version 3.3
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gtgagtgaga gccgctgtgc cgggcagggc ctggtggggg cactgcggac ctgggcacgg 60
acacgtgccc gggagactgc cgtggttctc gtacgggaca ccggaaccac cgacgacacg 120
gcgtcggtgg actacggaca gctggacgag tgggccagaa gcatcgcggt gaccctccga 180
cagcaactcg cgccgggggg acgggcactt ctgctgctgc cgtccggccc ggagttcacg 240
gccgcgtacc tcggctgcct gtacgcgggt ctggccgccg taccggcgcc gctgcccggg 300
gggcgccact tcgaacgccg ccgtgtcgcg gccatcgccg ccgacagcgg agccggcgtg 360
gtgctgaccg tcgcgggtga gaccgcctcc gtccacgact ggctgaccga gaccacggcc 420
ccggctactc gcgtcgtggc cgtggacgac cgggcggcgc tcggcgaccc ggcgcagtgg 480
gacgacccgg gcgtcgcgcc cgacgacgtg gctctcatcc agtacacctc gggctcgacc 540
ggcaacccca agggcgtggt cgtgacccac gccaacctgc tggcgaacgc gcggaatctc 600
gccgaggcct gcgagctgac cgccgccact cccatgggcg gctggctgcc catgtaccac 660
gacatggggc tcctgggcac gctgacaccg gccctgtacc tcggcaccac gtgcgtgctg 720
atgagctcca cggcattcat caaacggccg cacctgtggc tacggaccat cgaccggttc 780
ggcctggtct ggtcgtcggc tcccgacttc gcgtacgaca tgtgtctgaa gcgcgtcacc 840
gacgagcaga tcgccgggct ggacctgtcc cgctggcggt gggccggcaa cggcgcggag 900
cccatccggg cagccaccgt acgggccttc ggcgaacggt tcgcccggta cggcctgcgc 960
cccgaggcgc tcaccgccgg ctacgggctg gccgaggcca ccctgttcgt gtcgaggtcg 1020
caggggctgc acacggcacg agtcgccacc gccgccctcg aacgccacga attccgcctc 1080
gccgtacccg gcgaggcagc ccgggagatc gtcagctgcg gtcccgtcgg ccacttccgc 1140
gcccgcatcg tcgaacccgg cgggcaccgt gttctgccgc ccggccaggt cggcgagctg 1200
gtcctccagg gagccgccgt ctgcgccggc tactggcagg ccaaggagga gaccgagcag 1260
accttcggcc tcaccctcga cggcgaggac ggtcactggc tgcgcaccgg cgatctcgcc 1320
gccctgcacg aagggaatct ccacatcacc ggccgctgca aagaggccct ggtgatacga 1380
ggacgcaatc tgtacccgca ggacatcgag cacgaactcc gcctgcaaca cccggaactt 1440
gagagcgtcg gcgccgcgtt caccgtcccg gcggcacctg gcacgccggg cttgatggtg 1500
gtccacgaag tccgcacccc ggtccccgcc gacgaccacc cggccctggt cagcgccctg 1560
cgggggacga tcaaccgcga attcggactc gacgcccagg gcatcgccct ggtgagccgc 1620
ggcaccgtac tgcgtaccac cagcggcaag gtccgccggg gcgccatgcg tgacctctgc 1680
ctccgcgggg agctgaacat cgtccacgcg gacaagggct ggcacgccat cgccggcacg 1740
gccggagagg acatcgcccc cactgaccac gctccacatc cgcaccccgc gatgaacccg 1800
cccgaagcgg tcagcacgcc cagcgaggtc accgcgtgga tcaccggaca gatcgccgag 1860
ttcgtgaacg agacacccga ccggatcgcc ggtgacgcac ccctgaccga ccatggcctc 1920
gactccgtct ccggagttgc cctctgcgcg caggtcgagg accgctacgg gatcgaggtc 1980
gacccggagc tgctgtggag cgtccccaca ctcaacgagt tcgtccaggc actgatgccc 2040
cagttggccg accgcacctg aggggatccg cgagagatgg acatgcagtc gcagcgcctc 2100
ggcgtcaccg ccgcccaaca gagcgtctgg ctcgccggcc agctggcgga cgaccaccgc 2160
ctgtaccact gtgcggcgta cctgtcactc accgggtcca tcgacccgcg gacactcggc 2220
acggcggtcc ggcggaccct cgacgagacc gaggcgctgc gtacccggtt cgtaccgcag 2280
gacggggaac tgctgcagat cctcgaaccc ggtgccggac agctcctgct ggaagccgac 2340
ttctccggcg acccggaccc cgagcgggcg gcacacgact ggatgcacgc ggcgctcgcc 2400
gcaccggtcc gcctcgaccg cgccgggacc gccacccacg ccctgctcac cctcggcccg 2460
tcccgccacc tgctgtactt cggctaccac cacatcgcgc tcgacggcta cggtgccctg 2520
ctccacctgc gccgcctcgc ccacgtctac accgccctca gcaacgggga cgaccccggc 2580
ccctgcccgt tcggccccct ggccggtgtc ctcacggagg aggcggccta ccgtgactcc 2640
gacaaccatc ggcgcgacgg ggaattctgg acccggtccc tcgccggtgc ggacgaggcc 2700
cccgggctga gcgagcggga ggccggcgct ctcgccgtcc cgctgcgccg caccgtggag 2760
ctgtccggcg aacggacgga gaagctggcc gcctcggccg cggccactgg agctcgctgg 2820
tcgtcactgc tcgtcgccgc caccgccgcg ttcgtacgcc gccacgctgc cgccgacgac 2880
accgtcatcg gcctgcccgt caccgcccgg ctcaccgggc cggcgctgcg taccccgtgc 2940
atgctcgcca acgacgtgcc gctgcgcctc gacgcccggc tcgatgcccc gttcgccgcg 3000
ctccttgccg acaccacccg cgccgtcggc acgctggcgc gccaccagcg gttccgcggg 3060
gaagaactcc accggaacct ggggggcgtc ggccgcaccg cgggcctggc gcgggtcacc 3120
gtcaacgtcc tggcgtatgt cgacaacatc cggttcggcg actgccggg 3169
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aggtgatgag gtgcagcccg gtcaccccgt gcagcgcggg agccagcgac gccgggtccg 60
tgagatcgcc gccgacgacc tcgacccccg ccgggaactc cgcccgctcc gggtcgcggg 120
tcagcgcccg gacctgctcg ccacgctcca gcagctgctc gaccacgcgg cggccgaccg 180
ttcccgtggc tccggtcacc aggatcgtca tgaggatctc cttcgtcggt tccactccgt 240
ctgagcggca acacgaccaa ggtagggacc cttgcggaca gcttcggtcc tcaatcagcc 300
ccgtccggaa gggaattcag gcggagaact caggcggaga actcaccagg cgagctgggt 360
gatctcctcg gcgacgacgg cgcaggcgtc ggcggccgga tcgatcagcg ggaagtgccc 420
gacgccgccg agcagcgtca gcccgaccgt ctcgccggcc ttggccgccg cgtcgacgta 480
cgcctcggag acggcctggg gtacgacgat gtcctcgacg ccctgcacga tcgcggtggc 540
gatcccggtc ggcagcagca ccccggggtc ggcgtgcgcc gcccggtccg cgaatccggc 600
ctccccgccc aggagctgcg tgaccgcacc gccgcacacc ccgaggccca ccgccgccgc 660
gaagtccgcg atcggggcca gggcgacgac cccgcgcagc ggcggcgggg cgggcagccg 720
ccacggcgac ccctcgggca ggacgtgccg cgccgccgcc cagagcgcga ggtgcccgcc 780
cgcggagtgc ccggtgacca cgatccgccg cacatcggcg cccggcagcg cgcgggcggc 840
cagcacgggc atcgcgtcca gggccgcggc cacgtcgtcg aaggtctccg gccagcgccc 900
cgcgactggg cccgcgccgg cggccccggt gctctcctca ccgccgcgct gttgcgggat 960
gtcgtcgccg ccgcgccggt actccacgtt cgccacggcg aagccccggc gggccaggaa 1020
gtccgcgagc ggggagacat gggcccggtc gtacggggcc cgccaggcgc cgccgtgcag 1080
gaggacgacg agcggggcgg cggtgcggcc gtcgcgcggg gcgtagaagt cgacgacctg 1140
gtcgggatgg tccccgtagg cggcggtggc gtcgggggcg acgggaggat gggcgaacgc 1200
cgacgcgctc tccgcctcgt cccgatcacg cgccgcagcg tccggggaac ccggggaatc 1260
cgacatgccg ctcaaccgtc ctctgcccgt ggcctgtgcc tgttgccctg tgctctgtgc 1320
tctgtgcagg tgttctgtgc ctgcgtcagg tgcccaactg ggttgacctg cggggacggt 1380
accagcactc ggcaccgtcc ccgcccgccc gaacccctgt tgacgagctg tcaggcggcg 1440
ggttccccgg cgagcacctc ggcgaggatc cgcgccgccc gttccgcgtc ggcgaatccg 1500
acgtacagcg gggtgaagcc tctagaggat cccgagttgc cgtcgaggta gacgccgtcg 1560
tcgagcgcga agagctcgcg gcggtcggcc agcggatcgg cggcgtcgag cttcagcgcc 1620
cgctcgcgga gggcttccgt cgtggtcgtg gtcgcggtcg tggtctcaga catggctgcg 1680
cgccgtccac agctcgggga acacgttctt cgtggcgcgc ttctccagcc aggcgacccc 1740
ggcggaaccg ccggtgccgg tcttggagcc catcgctcga cgggtggcga ccaggtggtc 1800
gttgcgccag cgccacacca gctcgccgac gtcggtgagc agctcaccga ggcggaccag 1860
ctcgtcgttc tggtcgtcgg aggcgtagat ctccgcccac acggcctcga cctcgggcga 1920
cggctcgtac cgcttggtga ggtcccgggt ggtgacggcc tcggggacgg catacccgcg 1980
ccgggccagc agggcgaggg tctcgtcgta caggctgggc tccccgagcg ccttctccag 2040
ctcggcgtac acgcggggcg cgccccggtg cggcaccagc atggaggcgg acttgtcgcc 2100
gagcaggaac tccatccgcc ggtacatcgc ggactggaat cccgacccct cgccgaggga 2160
gccccggtag gagttgaact gggcgggggt gagtccggcc agcggccgcc aggactcgtt 2220
gagcgcctcc agctcgcgca gtgaacgctt gagggcgtcg cgcgcgacgg gtatgcggtc 2280
ctcgcgcagg gcgcgggtgg cggtctccca ctcatggacg atgaccgtga accacagctc 2340
catgacctgg gtggtgacca ggaaaaccat ctcgccggga tcgtccgagc gcaggtgctg 2400
gaggtgggtc agtacatccg cctggacata gtcctcgtac ggagtcgttc ccgcgaagtc 2460
caggtacggg gtgtccgcac cggtggcttc ggggtcgggg tggttggtcg acatttctgt 2520
ctcctcgaca cgagcttccg ggtagcggtc cgccccttcc gtgaggtgat ggagctccgg 2580
tcccctgctc gcatactaag ccggacccgc gcgacatggg cggcgggccc ggctcgggag 2640
tacgtccccg ggctcagccc aggatgtcgg cggcggtcgg cgaggagtcg cgcaggaacg 2700
tcgtgcagcg ctcgtgctcc tcggtctccc cgatcgaccc ggcggcccgg gccagggcgt 2760
gcagggcgcg caggaagccg cggttcggct cgtgctcgaa cgggacgggc ccgtggccct 2820
tccagccggc ccggcgcagg gagtcgaggc cgcggtggta gccggtacgg gcgtacgcgt 2880
aggactcgac gacccggccg ccctcgtacg cctcgtcggc gagctgcgcc caggcgagcg 2940
aggaggtggg gtacttcgcg gcgacctcgg cgggcggggt gccggcggcg agcagctcgc 3000
gcggctcggg gtcgtcgggg aggtgggtcg gggcggggcc cccgagcagg ttctcgtgga 3060
tggacat 3067
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gatccggcgg cttgcgcccg atgctagtcg cggttgatcg gcgatcgcag gtgcacgcgg 60
tcgatcttga cggctggcga gaggtgcggg gaggatctga ccgacgcggt ccacacgtgg 120
caccgcgatg ctgttgtggg ctggacaatc gtgccggttg gtaggatctg actgagtgac 180
caaaggaggc ggacatatga cagcgcagga cacccggacg accgggagtg acggtggcgg 240
ccggggcgcc acgtaccacg agagcccgac ctacggggag ctgctgcgcc tggaggacct 300
gctgaacgtc gcgcacctgc gcgacgcggc cgccccggtc ctcttccttg ccacgcacca 360
gtcggcggag atctggttcg gcatcgtgct gcgccacctg gaggaaatcc gcgcggccct 420
cacggacgac gacccggaca cggcactgca tctgctgccg cgactgccgg agatcttcga 480
actgctcgtc cgccacttcg acatgctggc cacgctgagt acggaggaat tcggcaagat 540
ccgcgcgggg ctgggcacgg cgagcggctt ccagtcggcg cagtaccggg agatcgagtt 600
cctgtgcggt ctgcgcgacc accgccacat ctccacaccg ggcttcacgg aaaccgaacg 660
tcggcgactg cgggaacggg cccgccagcc ctccgtggcg gaggcctacg acgccttccg 720
gacccgatgc gccaacggga aggacgcgga acggatcggg gaagcgctcc tgaggttcga 780
cgaacgggtc accgtctggc gcgcccgcca cgcggccctg gcggaacgct tcctgggccc 840
ccttgaaggg acggccggca ccgccggagc cgactacttg tggcgggtca cccggcacag 900
gctcttcccc ccggaggcgt ggggcgccgg ctgacggcac cgcccgatat cggaagctcg 960
tgtcgaggag acagaaatgt cgaccaacca ccccgacccc gaagccaccg gtgcggacac 1020
cccgtacctg gacttcgcgg gaacgactcc gtacgaggac tatgtccagg cggatgtact 1080
gacccacctc cagcacctgc gctcggacga tcccggcgag atggttttcc tggtcaccac 1140
ccaggtcatg gagctgtggt tcacggtcat cgtccatgag tgggagaccg ccacccgcgc 1200
cctgcgcgag gaccgcatac ccgtcgcgcg cgacgccctc aagcgttcac tgcgcgagct 1260
ggaggcgctc aacgagtcct ggcggccgct ggccggactc acccccgccc agttcaactc 1320
ctaccggggc tccctcggcg aggggtcggg attccagtcc gcgatgtacc ggcggatgga 1380
gttcctgctc ggcgacaagt ccgcctccat gctggtgccg caccggggcg cgccccgcgt 1440
gtacgccgag ctggagaagg cgctcgggga gcccagcctg tacgacgaga ccctcgccct 1500
gctggcccgg cgcgggtatg ccgtccccga ggccgtcacc acccgggacc tcaccaagcg 1560
gtacgagccg tcgcccgagg tcgaggccgt gtgggcggag atctacgcct ccgacgacca 1620
gaacgacgag ctggtccgcc tcggtgagct gctcaccgac gtcggcgagc tggtgtggcg 1680
ctggcgcaac gaccacctgg tcgccacccg tcgagcgatg ggctccaaga ccggcaccgg 1740
cggttccgcc ggggtcgcct ggctggagaa gcgcgccacg aagaacgtgt tccccgagct 1800
gtggacggcg cgcagccatg tctgagacca cgaccgcgac tctagacagg ccacgggcag 1860
aggacggttg agcggcatgt cggattcccc gggttccccg gacgctgcgg cgcgtgatcg 1920
ggacgaggcg gagagcgcgt cggcgttcgc ccatcctccc gtcgcccccg acgccaccgc 1980
cgcctacggg gaccatcccg accaggtcgt cgacttctac gccccgcgcg acggccgcac 2040
cgccgccccg ctcgtcgtcc tcctgcacgg cggcgcctgg cgggccccgt acgaccgggc 2100
ccatgtctcc ccgctcgcgg acttcctggc ccgccggggc ttcgccgtgg cgaacgtgga 2160
gtaccggcgc ggcggcgacg acatcccgca acagcgcggc ggtgaggaga gcaccggggc 2220
cgccggcgcg ggcccagtcg cggggcgctg gccggagacc ttcgacgacg tggccgcggc 2280
cctggacgcg atgcccgtgc tggccgcccg cgcgctgccg ggcgccgatg tgcggcggat 2340
cgtggtcacc gggcactccg cgggcgggca cctcgcgctc tgggcggcgg cgcggcacgt 2400
cctgcccgag gggtcgccgt ggcggctgcc cgccccgccg ccgctgcgcg gggtcgtcgc 2460
cctggccccg atcgcggact tcgcggcggc ggtgggcctc ggggtgtgcg gcggtgcggt 2520
cacgcagctc ctgggcgggg aggccggatt cgcggaccgg gcggcgcacg ccgaccccgg 2580
ggtgctgctg ccgaccggga tcgccaccgc gatcgtgcag ggcgtcgagg acatcgtcgt 2640
accccaggcc gtctccgagg cgtacgtcga cgcggcggcc aaggccggcg agacggtcgg 2700
gctgacgctg ctcggcggcg tcgggcactt cccgctgatc gatccggccg ccgacgcctg 2760
cgccgtcgtc gccgaggaga tcacccagct cgcctggtga tctagactaa gcttgcggcc 2820
gcccggctgc agcccgtacc tctagtagag tcgaggggga ac 2862
Claims (1)
1.一种产达托霉素的玫瑰孢链霉菌（Streptomyces roseosporus）L31 ，其保藏编号为CGMCC No：14233。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002059322A3 (en) * | 2000-10-17 | 2003-10-30 | Cubist Pharmaceuticlas Inc | Compositions and methods relating to the daptomycin biosynthetic gene cluster |
| CN102146414A (zh) * | 2010-07-26 | 2011-08-10 | 天津大学 | 玫瑰孢链霉菌基因工程菌的构建方法及其应用 |
| CN102485902A (zh) * | 2010-12-06 | 2012-06-06 | 北大方正集团有限公司 | 一种发酵生产达托霉素的方法 |
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| WO2002059322A3 (en) * | 2000-10-17 | 2003-10-30 | Cubist Pharmaceuticlas Inc | Compositions and methods relating to the daptomycin biosynthetic gene cluster |
| CN102146414A (zh) * | 2010-07-26 | 2011-08-10 | 天津大学 | 玫瑰孢链霉菌基因工程菌的构建方法及其应用 |
| CN102485902A (zh) * | 2010-12-06 | 2012-06-06 | 北大方正集团有限公司 | 一种发酵生产达托霉素的方法 |
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| Title |
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| Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry;Vivian Miao 等;《Microbiology》;20051231;第1507-1523页 * |
| 达托霉素生物合成研究进展;谢祥茂 等;《微生物学通报》;20131017;第1754-1764页 * |
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