CN109266662A - 一组生物合成创新霉素或开环创新霉素的基因簇 - Google Patents

Abstract

本发明涉及一种创新霉素和/或开环创新霉素的生物合成的基因簇及相关所需基因，其特征在于，所述的基因簇序列核苷酸如SEQ ID No.1所示，其中依次包含cxnA、cxnB、cxnC、cxnD、cxnE、cxnF、cxnT、cxnR、trpRS九个关键功能基因，以及在创新霉素分子中引入S元素所必需的thiG、moeZ基因，其核苷酸序列分别为如SEQ ID No.2～12所示。

Description

一组生物合成创新霉素或开环创新霉素的基因簇

技术领域

本发明属于医药技术领域，具体而言，涉及一组合成创新霉素的基因簇。

背景技术

创新霉素(chuangxinmycin，简称CM，化学结构见图1)是20世纪70年代中国医学科学院医药生物技术研究所(原名：中国医学科学院抗菌素研究所)首先发现的一个具有含氮、含硫杂环新骨架的抗生素，由从我国山东济南土壤中分离的1株游动放线菌(Actinoplanes tsinanensis CPCC 200056)产生。在临床上，CM对大肠杆菌等细菌感染所引起的败血症、尿路感染、胆道感染和婴儿腹泻等有一定疗效，临床试用的有效率达77.86％[1]。

由于CM具有新颖的骨架结构和良好的抑菌活性，国内外学者对其进行了多方面研究。在化学方面，已确定CM的绝对构型[2]并建立了CM化学全合成路线，获得了一系列CM衍生物[3-5]；在生物学活性方面，已证实CM的抗菌作用是通过选择性抑制色氨酸tRNA合成酶活性实现的[6]；在生物合成方面，戚天庆等从CM产生菌中分离纯化了一种可能参与催化CM生物合成的吲哚丙酮酸甲基转移酶[7]，左利杰等从CM产生菌中分离得到3-去甲创新霉素(demethyl-chuangxinmycin,DCM,图1)，但目前还不清楚DCM是CM生物合成过程中出现的中间产物还是支路产物[8]；许津等利用同位素标记实验推测CM分子中的硫原子可能来源于半胱氨酸(Cys)的巯基[9]，以及周锡漳等探讨了维生素B12在CM生物合成中的作用[10]。但是到目前为止，CM的生物合成基因簇尚未报道，关于CM中硫原子的掺入机制以及C-S键(碳硫键)的形成等生物合成机制仍为未知。因此，CM生物合成基因簇的解析将阐明其生物合成机制及调控机制，为利用遗传操作提高其产量，或者获得新结构衍生物用于新药发现奠定基础。

众所周知，在微生物次级代谢产物的生物合成基因簇中，其抗性基因和生物合成基因往往共同存在以保护产生菌免于其产生的抗生素的损害。吲哚霉素(indolmycin)和CM的抗菌机制相同，也属于色氨酸tRNA合成酶抑制剂。2015年，Du等在吲哚霉素产生菌全基因组范围内扫描色氨酸tRNA合成酶的同源基因从而发现吲哚霉素的生物合成基因簇[11]。我们推测，在CM生物合成基因簇内可能存在色氨酸tRNA合成酶基因。

另一方面，创新霉素结构中一个显著特征是其含有S元素，S的掺入以及C-S键的形成应为其生物合成的主要步骤。到目前为止，次级代谢产物生物合成过程中S的掺入机制仍所知甚少。2014年，Sasaki等报道了Amycolatopsis orientalis subsp.vinearia BA-07585中次级代谢产物BE-7585A生物合成中S的掺入机制[12]：利用原核生物初级代谢中维生素B1(thiamine)、钼蝶呤、半胱氨酸/甲硫氨酸等的硫原子掺入机制，即硫载体蛋白(sulfur-carrier protein,SCP)、SCP活化蛋白等共同完成。在BE-7585A生物合成基因簇内存在催化维生素B1中噻唑合成酶ThiG[13]的同源蛋白BexX，但该基因簇中不含有SCP；在基因组中存在几个SCP的同源蛋白(ThiS,MoaD,CysO和MoaD2)，但仅有唯一的SCP活化蛋白ThiF的同源蛋白MoeZ(C-端还含有硫氰酸酶结构域，催化活化的SCP的硫醇化)。Sasaki等通过实验证实A.orientalis中此三个元件(BexX，SCP，MoeZ)负责BE-7585A中2-硫糖结构的S的掺入。

发明内容

本发明首先涉及创新霉素生物合成的基因簇，所述的基因簇依次包含cxnA、cxnB、cxnC、cxnD、cxnE、cxnF、cxnT、cxnR、trpRS九个关键功能基因，以及在创新霉素分子中引入S元素所必需的thiG、moeZ基因，九个关键基因和thiG、moeZ基因的核苷酸序列分别为如SEQID No.2～12所示，包含cxnA、cxnB、cxnC、cxnD、cxnE、cxnF、cxnT、cxnR、trpRS九个关键功能基因的基因片段序列如SEQ ID No.1所示，

本发明还涉及创新霉素生物合成基因簇中的各个功能蛋白：

CxnA为依赖于维生素B12的自由基S-腺苷甲硫氨酸家族的C-甲基转移酶，其功能为催化吲哚丙酮酸甲基化生成3-甲基吲哚丙酮酸；

CxnB为氨基转移酶，其功能为将创新霉素的生物合成前体色氨酸(L-Trp)中的氨基转变为酮基，形成吲哚丙酮酸；

CxnC为还原酶，其功能为将3-甲基吲哚丙酮酸的酮基还原为羟基，形成3-吲哚-2-羟基丁酸；

CxnD为细胞色素P450酶，其功能为催化CM的开环结构(secochuangxinmycin,SCM)中的S原子和C原子形成C-S键，形成CM；

CxnE为硫载体蛋白，在JAMM金属蛋白酶家族的CxnF的作用下，C-末端10个氨基酸残基被水解露出双甘氨酸基序，而后先在SCP活化蛋白MoeZ(N-端为类似于SCP活化蛋白ThiF的结构域)作用下生成腺苷酰化的SCP，再在MoeZ的C-端硫氰酸酶结构域的作用下形成硫醇化的SCP，即为硫原子的供体；

CxnF为JAMM金属蛋白酶家族成员，其功能为酶切CxnE的C-末端10个氨基酸残基，露出双甘氨酸基序；

CxnR为转录调控蛋白，其功能为调节所述基因簇的表达；

CxnT为创新霉素的转运蛋白；

TrpRS为色氨酸tRNA合成酶，是创新霉素的自身抗性基因；

ThiG为噻唑合成酶，其功能为将硫醇化SCP中的S原子转移至还原酶CxnC催化形成的3-吲哚-2-羟基丁酸的羟基，得到3-吲哚-2-巯基丁酸(即CxnD的底物，开环创新霉素)；

MoeZ为SCP活化蛋白，其作用为活化SCP蛋白CxnE，使其硫醇化形成硫代羧酸酯。

本发明还涉及所述的基因簇中的各个基因thiG、cxnA、cxnB、cxnC、cxnD、cxnE、cxnF、cxnT、cxnR、trpRS、moeZ编码的蛋白质，其氨基酸序列分别为Seq ID No.13～23所示。

本发明还涉及一种开环创新霉素SCM，其结构式如下式所示

本发明还涉及所述的开环创新霉素SCM的生物合成基因簇，包括cxnA、cxnB、cxnC、cxnE、cxnF、cxnT、cxnR、trpRS及thiG、moeZ十个功能基因。

本发明还涉及包括所述的开环创新霉素或创新霉素基因簇的重组载体，优选的，所述的重组载体为包含酵母菌元件(ARSH4/CEN6复制子和TRP1筛选标记)、大肠杆菌元件(pUC ori复制子)和链霉菌元件(φC31整合酶及其整合位点attP、DNA接合转移起始位点oriT)的重组载体，最优选的，所述的重组载体为pCAP01。

本发明还涉及转化了所述的重组载体的宿主，优选的，所述的宿主为酵母、大肠杆菌或链霉菌，最优选的，所述的宿主为链霉菌。

本发明还涉及通过生物发酵生产CM或SCM的方法，其步骤包括：

(1)将所述CM或SCM的生物合成基因簇克隆至目标宿主，

(2)发酵所述目标宿主并从发酵液中提取并纯化所述CM或SCM。

本发明还涉及所述的创新霉素生物合成基因簇或所述的开环创新霉素生物合成基因簇和/或其编码蛋白在催化合成创新霉素、开环创新霉素或其类似物中的应用。

本发明还涉及所述的cxnR基因或其编码的CxnR蛋白在合成创新霉素、开环创新霉素中的应用。

本发明还涉及一种提高创新霉素、开环创新霉素产量的方法，其特征在于，在生产菌株中过表达所述cxnR基因，所述的过表达的方法为，将高表达所述cxnR基因的重组载体转入创新霉素、开环创新霉素的生产菌株中，优选的，所述的重组载体为质粒pSET152，所述的生产菌株为来自中国药学微生物菌种保藏管理中心的野生型菌株Actinoplanestsinanensis CPCC 200056(China Pharmaceutical Culture Collection)。

本发明还涉及所述的cxnD基因或其编码的CxnD蛋白在以开环创新霉素为底物合成创新霉素中的应用。

本发明还涉及一种合成创新霉素的方法，其特征在于，以开环创新霉素为底物，发酵转化了所述cxnD基因的宿主，所述的宿主不包含所述创新霉素合成基因簇的其他基因，优选的，所述的宿主为链霉菌。

附图说明

图1、创新霉素(CM)、去甲创新霉素(DCM)及开环创新霉素(SCM)的结构示意图。

图2、创新霉素生物合成基因簇及其相邻基因：thiG(ats3059)与moeZ(ats6133)位于基因簇外，但推测参与CM的生物合成；左臂和右臂为DNA Assembler方法中的捕捉臂；“deleted”为阻断菌株M1146/pCAP-CM(ΔcxnA～F)中删除基因部分。

图3、pL-CxnR的质粒图和酶切鉴定结果：

3A、pL-CxnR质粒示意图；

3B、pL-CxnR质粒酶切鉴定结果：Lane 1～4：NdeI和XbaI双酶切鉴定pL-CxnR，预计大小6kb和0.95kb；Lane M：1kb plus DNA ladder(10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,500,1000,800,500,300bp)。

图4、重组菌株的PCR验证：Lane M：1kb plus DNA ladder(10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,500,1000,800,500,300bp)；WT：CPCC 200056；-：PCR的阴性对照。

图5、TLC检测各菌株中CM的产量：1～4：200056/pSET152的4个单克隆；5～8：200056/pL-CxnR的4个单克隆；WT：CPCC 200056；CM：创新霉素标准品。

图6、HPLC检测各菌株中CM的产量：

6A、200056/pSET152和200056/pL-CxnR其中一个单克隆发酵产物的HPLC图；

6B、CM产量的HPLC定量比较(200056/pSET152与200056/pL-CxnR分别为4个和8个单克隆的统计结果)。

图7、利用DNA Assembler构建质粒pCAP01-CM及相关验证：

7A、pCAP01-CM的构建示意图；

7B、从E.coli DH5α中提取的质粒pCAP01-CM进行PCR验证。Lane 1～2：扩增左臂片段，预计大小2265bp；Lane 3～4：扩增右臂片段，预计大小2225bp；Lane 5～6：扩增片段1，预计大小4003bp；Lane 7～8：扩增片段2，预计大小4101bp；Lane 9～10：扩增片段3，预计大小4049bp；Lane 11～12：扩增片段4，预计大小4388bp；Lane M：1kb plus DNA ladder(10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,500,1000,800,500,300bp)；

7C、从E.coli DH5α中提取的质粒pCAP01-CM进行限制性内切酶验证。Lane 1～2：SpeI酶切(28242bp)；Lane 3～4：NdeI酶切(28242bp)；Lane 5～6：EcoRI酶切(23132，4336bp)；Lane 7～8：KpnI酶切(10834，10483，3340，1884，1601bp)；Lane 9～10：XhoI酶切(12553，6319，3031，2415，1002，951，714，444，399，237，141bp)；Lane M：1kb DNA ladder(10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,000bp)。

图8、重组菌株的PCR验证：

8A、利用φC31整合位点通用引物(pSET152与attB_Streptomyces)进行验证。预计大小1.6kb；Lane M：1kb plus DNA ladder(10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,500,1000,800,500,300bp)；Lane 1,13,14：S.coelicolor M1146；Lane 2～6：M1146/pCAP-CM(ΔcxnD)的5个单克隆；Lane 7～11：M1146/pSET152的5个单克隆；Lane 12：PCR阴性对照；Lane 15～19：M1146/pCAP-CM(ΔcxnA～F)的5个单克隆；

8B、利用在删除序列的上下游设计的引物(CM-23K-1和CM-23K-2)进行验证。LaneM：1kb DNA ladder(10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,000bp)；Lane1：PCR阴性对照；Lane 2：S.coelicolor M1146；Lane 3～7：M1146/pCAP-CM的5个单克隆，预计大小5,851bp；Lane 8～15：M1146/pCAP-CM(ΔcxnA～F)的8个单克隆，预计大小741bp；

8C、利用φBT1整合位点设计引物(BT1-M1146-F1与BT1-M1146-R1)进行验证。预计大小1.0kb；Lane M：1kb plus DNA ladder(10,000,8,000,6,000,5,000,4,000,3,000,2,000,1,500,1000,800,500,300bp)；Lane 1～8：M1146/pCAP-CM(ΔcxnD)/pIJ10500的8个单克隆；Lane 9～20：M1146/pCAP-CM的12个单克隆；Lane 21：S.coelicolor M1146；Lane 22：M1146/pCAP-CM(ΔcxnD)。

图9、LC-MS分析异源表达产物：

9A、各菌株发酵产物的HPLC分析；I、野生型菌株发酵产物的HPLC分析；II、M1146/pSET152发酵产物的HPLC分析；III、M1146/pCAP-CM发酵产物的HPLC分析；IV、M1146/pCAP-CM(ΔcxnA～F)发酵产物的HPLC分析；V、M1146/pCAP-CM(ΔcxnD)发酵产物的HPLC分析；

9B、图A中各个峰的MS分析图；依次为1(即CM)的MS分析图、2(即DCM)的MS分析图、3(即SCM)的MS分析图；C、CM、DCM和SCM的紫外吸收光谱；

9C、CM、DCM和SCM的紫外吸收光谱。

图10、CM与SCM的二级高分辨质谱图：

10A、创新霉素的二级高分辨质谱图；

10B、开环创新霉素的二级高分辨质谱图。

图11、CM生物合成过程示意图：

11A、硫载体蛋白的活化；

11B、CM生物合成途径分析。

具体实施方式

材料与方法

1、菌株、质粒和培养方法

野生型菌株Actinoplanes tsinanensis CPCC 200056来自中国药学微生物菌种保藏管理中心(China Pharmaceutical Culture Collection)，其培养和固体发酵均使用ISP2培养基(BD NO.277010)，接合转移使用three medium 65培养基[23]；Streptomycescoelicolor M1146及其相关链霉菌菌株的培养和接合转移均使用MS培养基[24]，固体发酵使用ISP2培养基；培养和发酵均于28℃恒温培养7d；所有菌株提取基因组DNA时都使用液体φ培养基[25]培养。Saccharomyces cerevisiae VL6-48是用来克隆生物合成基因簇的宿主菌，使用YPAD培养基培养[20]，利用DNA Assembler进行克隆时使用SD-Trp培养基(SigmaNO.630411和630413)进行筛选，培养温度30℃。Escherichia coli DH5α作为通用的大肠杆菌克隆宿主[26]，E.coli ET12567/pUZ8002是用于在大肠杆菌及链霉菌之间进行接合转移的宿主[27]，二者使用LB培养基37℃恒温培养。当需要用抗生素时，它们的工作浓度如下：阿普霉素(apramycin,Am,50μg/ml)，潮霉素(hygromycin B,Hyg,200μg/ml)，卡那霉素(kanamycin,Km,50μg/ml)，氯霉素(chloramphenicol,Cm,30μg/ml)和萘啶酮酸(nalidixicacid,ND,25μg/ml)。本文中所有的菌株和质粒见表1，引物见表2。

表1、菌株和质粒

Am^r,apramycin resistance；Km^r,kanamycin resistance；Cm^r,chloramphenicolresistance；Hyg^r,hygromycin B resistance

表2、引物

Restriction endonuclease recognition sequences introduced by theoligonucleotides are bold.

2、提取高质量的Actinoplanes tsinanensis CPCC 200056基因组

将A.tsinanensis CPCC 200056接种于100ml的φ培养基中，28℃，230rpm振荡培养24h。5,000rpm离心10min收集15ml菌体，并用STE缓冲液(10mM Tris-HCl,1mM EDTA,pH8.0)洗涤菌体一次。用5ml STE缓冲液重悬菌体，加入终浓度为5mg/ml的溶菌酶，在37℃恒温水浴中孵育30min。然后加2ml 2％SDS轻轻混匀后，加入等体积的酚：氯仿：异戊醇(25:24:1)，在室温下轻轻摇匀10min，5,000rpm,4℃离心10min。将上清液转移到新的离心管中。加RNase A至终浓度15μg/ml，在37℃恒温水浴中孵育30min，然后加入等体积的酚：氯仿：异戊醇(25:24:1)，在室温下轻轻摇匀10min，5,000rpm,4℃离心10min。将上清液转移到新的离心管中。重复抽提1～2次，直至界面没有白色变性蛋白的存在。加入等体积的氯仿抽提一次以除去残留的酚，5,000rpm,4℃离心10min。

将上清液转移到新的离心管中，加入1/20体积的5M NaCl和等体积异丙醇，轻轻地充分混匀至DNA沉淀。用灭菌的玻璃棒缠绕DNA，将其移至新的离心管中，70％的乙醇洗涤一至两次，室温晾干后溶于500μl ddH₂O，保存于4℃备用。经电泳检测符合送样要求后，送样测序。

3、菌株发酵及产物的TLC、HPLC和LC-MS分析

本文中所有菌株都采用ISP2培养基进行固体发酵，在28℃恒温摇床间中倒置培养7d，然后将每个ISP2平板培养物切碎成1cm×1cm的小方块，用2倍体积乙酸乙酯萃取48h，最后将乙酸乙酯萃取液浓缩至相同体积(250～500μl)，获得固体发酵产物。

硅胶板TLC分析的点样量为5μl，展开剂系统为乙酸乙酯-正己烷-二氯甲烷-冰醋酸，9:7:6:0.2，v/v，分别在254和365nm观察。

将最终的乙酸乙酯萃取液过滤，滤膜为0.22μm有机相专用，滤液进行HPLC分析。HPLC条件包括：1、XSelect CSH^TM C₁₈色谱柱(4.6×150mm,5μm,Waters,Ireland)或EclipsePlus C18色谱柱(4.6×150mm,5μm,Agilent,America)或CAPCELL PAK ADME色谱柱(4.6×250mm,5μm,SHISEIDO,Japan)；2、流动相A：0.1％CH₃COOH，流动相B：乙腈；3、梯度洗脱:在30min内，0.1％CH₃COOH-MeCN从85(A):15(B)逐渐变换到0(A):100(B)；4、流速设定为1.0ml/min；5、检测波长设定为254nm；6、柱温和检测池温度均为室温(25℃)。

进行LC-MS分析时，HPLC条件相同，将流速改为0.8ml/min。使用的仪器为1290(LC)-1956single quadrupole MS(Agilent,America)或1100(LC)-6300MSD Trap MS(Agilent,America)；MS条件为：电喷雾(ESI)离子源，负离子全扫描模式，电喷雾电压：4.5kV；加热毛细管温度：325℃；载气：N₂。

将化学合成的CM标准品和利用HPLC制备纯化的SCM进行HRESIMS和HRESIMS-MS分析，使用的仪器为QSTARTM Elite LC/MS/MS system(Applied Biosystems/MSD Sciex,Singapore)，配有ESI源，采用负离子TOF扫描模式。

4、Actinoplanes tsinanensis CPCC 200056与E.coli ET12567/pUZ8002之间的接合转移

借鉴文献报道的Actinoplanes friuliensis的接合转移方法[23]建立A.tsinanensis CPCC 200056的遗传操作系统。A.tsinanensis CPCC 200056在ISP2平板上于28℃培养7d，然后将培养好的菌体铲下一个大小约1cm×1cm的菌块接种至100ml TSB液体培养基中，28℃，220rpm，振荡培养约96h；然后以10％转接至50ml TSB液体培养基中，28℃，220rpm，振荡培养约16h；再以20％转接至50ml TSB液体培养基中，28℃，220rpm，振荡培养约1～5h，然后离心收集菌丝体(5,000×g,10min)，重悬于8～10ml TSB中。过夜培养的15ml大肠杆菌供体菌用15ml LB洗涤2次，最后重悬于1～2ml TSB中。将200μl大肠杆菌供体菌与200μl Actinoplanes tsinanensis CPCC 200056的菌丝体均匀混合后(二者的浓度均接近于10⁸cells/ml)，涂布在含有20mM MgCl₂的three medium 65平板[23]，放入28℃恒温摇床间中倒置培养约16～18h后，向每块平板表面覆盖3ml含有萘啶酸(ND,终浓度为25μg/ml)和阿普霉素(Am,终浓度为50μg/ml)的水，在超净台内吹干后放入28℃继续培养，大约3～5d以后挑取接合子，在ISP2(含50μg/ml Am)平板划线扩大培养，约7d后将菌株接种于5ml的φ培养基，28℃，220rpm振荡培养24～48h，利用Magen公司的细菌DNA小量提取试剂盒提取基因组总DNA，然后进行PCR验证。

5、DNA Assembler获取CM生物合成基因簇

Saccharomyces cerevisiae VL6-48在固体YPAD培养基上进行活化，30℃培养2d，挑取单克隆，接种至3ml液体YPAD培养基中，30℃，250rpm，振荡培养过夜。次日转接至50ml液体YPAD培养基中，调节OD₆₀₀接近于0.2，30℃，250rpm，振荡至OD₆₀₀接近于0.8，离心收集下层酵母菌体(4℃，4,000×g,10min)，用50ml预冷的ddH₂O洗涤一次，再次离心收集菌体，加1ml预冷的ddH₂O重悬菌体，而后转移至已灭菌的EP管中，离心收集菌体(4℃，4,000×g,1min)，加1ml预冷的1M山梨醇洗涤菌体，重复3次，最后将菌体重悬于200μl的1M山梨醇中，50μl/管进行分装，而后准备进行电转化。

准备用于电转化的DNA片段：捕捉载体pCAP-CM-LR先用EcoRI进行线性化处理，回收后用CIAP进行5’端脱磷处理，最后得到线性DNA片段；其他四个片段通过PCR获得高质量的DNA片段；相邻两个片段间的重叠区为100～800bp；将500-600ng的每个片段(大小约4K)和2μg的线性载体片段混匀，加乙醇进行沉淀，最后溶于4μl ddH₂O。

将4μl DNA片段与50μl酵母感受态细胞混匀，加入预冷的电击杯中进行电转化(电压：1.5KV)，而后立即加入1ml 30℃预热的YPAD液体培养基，30℃，250rpm，振荡培养约1h。离心(17,000×g,30s)弃上清，加1ml室温的1M山梨醇洗涤菌体重复3次，最后将菌体重悬于1ml 1M山梨醇中。分别取100μl、300μl和600μl菌液均匀涂布在SD-Trp平板，在30℃培养箱中倒置培养约2～3d后，可见克隆子产生。

挑取单克隆在SD-Trp平板上划方格，30℃培养2d，进行菌落PCR验证。验证正确的单克隆接种于3ml SD-Trp液体培养基中，30℃，250rpm，振荡培养约18h，离心收集菌体后，加入蜗牛酶(2U/μl)10μl，37℃温育30min～1h，离心收集原生质体，而后提取质粒转化E.coli DH5α，挑选单克隆，提取质粒进行PCR和酶切验证。

实施例1、创新霉素合成基因簇的生物信息学分析

我们首先对CM产生菌济南游动放线菌(Actinoplanes tsinanensis CPCC200056)进行DNA提取，利用全新三代测序Pacbio RSII平台结合二代测序Illumina Hiseq4000平台对其全基因组DNA进行测序，拼接组装获得其基因组精细图(该高通量测序工作由北京华大基因(Beijing Genomics Institute)完成)。A.tsinanensis CPCC 200056的基因组包含一个大小为7,685,618bp的线性基因组和一个13,534bp的环状质粒，G+C含量分别为70.3％和69.0％。经基因注释分析后发现，基因组分析含有7,060个编码基因，其中7,041个位于染色体上、19个位于环状质粒上。

对CM产生菌A.tsinanensis CPCC 200056基因组进行蛋白序列同源分析发现，在全基因组中存在唯一的ThiG(噻唑合成酶)同源蛋白(Ats3059)、唯一的MoeZ同源蛋白(Ats6133，SCP活化蛋白)和几个SCP同源蛋白(Ats3085、Ats3815、Ats4181、Ats4502、Ats4708)，而其中ats4181与色氨酸tRNA合成酶(TrpRS，吲哚霉素生物合成基因簇中Ind0亦为TrpRS)的同源基因(ats4186)相邻，在此附近还有吲哚霉素生物合成基因簇中存在的色氨酸氨基转移酶(Ind8)的同源基因(ats4179)，因此初步将CM的生物合成基因簇定位于此，命名为cxn基因簇(图2)。分析cxn中的序列：cxnA编码依赖于维生素B12的自由基S-腺苷甲硫氨酸家族的C-甲基转移酶；cxnB编码色氨酸氨基转移酶，与indolmycin生物合成中的ind8[11]及thienodolin生物合成中的thnJ[14]同源；cxnC与panE、apbA同源，编码2-脱氢泛解酸-2-还原酶；cxnD编码细胞色素P450类的氧化还原酶；cxnE编码SCP；cxnF编码未知功能蛋白，与thienodolin生物合成中的thnF[15]同源，分析三维结构时发现其含有MPN结构域，属于JAMM(JAB1/MPN/Mov34)金属蛋白酶家族，并且含有保守的JAMM基序(JAMM motif,EX_nHS/THX₇SX₂D，X代表任意氨基酸)；cxnT编码转运蛋白；cxnR编码LysR家族转录调控因子；trpRS编码色氨酸tRNA合成酶(表3)。

表3、创新霉素生物合成基因簇及功能

ats4177功能分析：

Ats4177通过BLASTP未找到已知功能的同源蛋白，但通过在线的HHpred对三维结构相似性进行分析时发现与分子伴侣PqqD具有很高的同源性，PqqD可以与PqqE(编码自由基S-腺苷甲硫氨酸家族的酶)相互作用从而参与或影响其功能，因此推测Ats4177可能通过与CxnA(为依赖于维生素B12的自由基S-腺苷甲硫氨酸家族的C-甲基转移酶)相互作用参与甲基化过程。

ats4183(cxnF)功能分析：

通过蛋白序列比对并未发现与之序列相似的已知功能蛋白，但分析此蛋白的三维结构时发现其含有MPN结构域，属于JAMM(JAB1/MPN/Mov34)金属蛋白酶家族。JAMM蛋白可以分为两类：一类为JAMM/MPN⁺亚家族，是金属蛋白酶，具有催化活性，并且在活性中心含有保守的JAMM基序(JAMM motif,EXnHS/THX7SX2D，X代表任意氨基酸)；另一类为JAMM/MPN^-亚家族，缺少催化活性，只作为多亚基复合物的一个组分。有报道发现有一些JAMM蛋白酶参与硫转运途径，比如萤光假单胞菌(Pseudomonas fluorescens)中合成含硫的thioquinolobactin(一种铁载体，siderophore)需要QbsE(SCP)的参与，但QbsE蛋白在其C-端的双甘氨酸基序(GG，diglycine motif)后还有两个氨基酸残基(CF)，即以前体形式存在，而JAMM蛋白酶QbsD可以将QbsE前体蛋白中的这两个氨基酸残基水解掉以利于SCP激活蛋白(MoeZ)进行后续的腺苷酰化，实现硫转运功能。将CxnF与已知的JAMM/MPN⁺亚家族蛋白(包括QbsD)进行序列比对发现CxnF中也含有保守的JAMM基序，而在cxn基因簇中SCP(cxnE)的C末端在双甘氨酸基序(diglycine motif)后还有10个氨基酸残基，因此我们推测CxnF的功能是水解CxnE的C末端的10个氨基酸残基，释放双甘氨酸基序以利于进行腺苷酰化，从而激活CxnE。

实施例2、调节基因cxnR的功能分析

在CM生物合成基因簇内存在一个可能的途径特异性调节基因cxnR，基因全长942bp，编码313个氨基酸。将CxnR的蛋白序列在GenBank中进行BLASTP分析，发现与Amycolatopsis lurida NRRL 2430中的LysR家族转录调控因子同源，一致性为37％；与Streptomyces hygroscopicus var.ascomyceticus ATCC 14891中安莎霉素生物合成的正调控蛋白FkbR1具有35％的一致性[16]。在产生菌对此调节基因进行过表达，分析创新霉素的产量，初步判断此基因簇是否负责创新霉素的生物合成。

载体pL646[17]来源于整合型质粒pSET152，其不含链霉菌复制子，含有φC31attP位点，可以整合至链霉菌基因组中的attB位点，在多克隆位点上游含有强启动子ermE*p和tuf1基因的SD序列。克隆cxnR的编码区至pL646的NdeI和XbaI位点，经酶切鉴定正确后得到重组质粒pL-CxnR(图3)。

CM的产生菌济南游动放线菌属于稀有放线菌，能形成孢囊，孢囊孢子微游动，在ISP2固体培养基上于28℃培养7d时，孢子在孢囊内，故而不适合采用传统的接合转移方法进行遗传操作。经调研和实验，成功建立了A.tsinanensis CPCC 200056的遗传操作系统(见材料与方法部分)。将过表达质粒pL-CxnR和对照质粒pSET152通过转化导入甲基化缺失的E.coli ET12567/pUZ8002菌株中，然后通过菌丝体接合转移导入A.tsinanensis CPCC200056中，从而获得过表达菌株200056/pL-CxnR和对照菌株200056/pSET152。提取重组菌株的基因组DNA，进行PCR验证。验证引物(pSET152与attB_Streptomyces)一端在基因组上，一端在质粒上，预计大小约1.6kb，如图4所示。

将验证正确的过表达菌株200056/pL-CxnR、对照菌株200056/pSET152和野生型菌株CPCC200056利用ISP2培养基进行固体发酵，将新鲜孢子悬液作为种子涂布接种于ISP2平板(直径9cm，含25ml培养基)，每块ISP2平板的接种量一致，约5×10⁵个孢子，而后在28℃恒温摇床间中倒置培养7d，然后将每个ISP2平板培养物切碎成小方块，用2倍体积乙酸乙酯萃取48h，最后将乙酸乙酯萃取液浓缩至相同体积(250～500μl)，获得固体发酵产物。首先对各发酵产物进行TLC检测分析(图5)，结果发现对照菌株200056/pSET152与野生型菌株CPCC200056中的CM条带灰度相当，而过表达菌株200056/pL-CxnR中的CM条带则明显加深，提示CM产量有所提高。

为了进一步定量分析各菌株发酵产物中的CM产量，我们进行了HPLC分析。利用峰面积进行相对定量分析(图6)，结果表明200056/pL-CxnR中的CM产量比200056/pSET152中的CM产量提高了98％，此结果表明CxnR参与了CM生物合成的调控，可能为其途径特异性正调控基因，提示其所在的基因簇是CM生物合成基因簇。

实施例3、CM生物合成基因簇及cxnD基因的功能分析

为了进一步确证CM的生物合成基因簇，我们采用近年来发展起来的克隆大片段的新兴技术─DNA Assembler[18]克隆分析的CM生物合成基因簇，然后导入S.coelicolorM1146[19]中进行异源表达分析。DNA Assembler是利用酵母菌中高效的同源重组机制来一步获得化合物的生物合成基因簇。本工作中将基因簇左右两端约2kb片段作为捕捉臂插入载体pCAP01[20]中即得到pCAP-CM-LR，其中pCAP01包含酵母菌元件(ARSH4/CEN6复制子和TRP1筛选标记)、大肠杆菌元件(pUC ori复制子)和链霉菌元件(φC31整合酶及其整合位点attP、DNA接合转移起始位点oriT)使其可在三种菌之间穿梭。将含有CM生物合成基因簇(ats4175-4190)的序列分成4个片段设计引物，通过高保真DNA聚合酶进行PCR得到DNA片段后，与线性化的捕捉载体(pCAP-CM-LR)一同通过电转化导入酿酒酵母中，利用同源重组进行拼接(相邻两片段之间均有一定的重叠)，得到含有CM生物合成基因簇的重组质粒pCAP01-CM(图7A)。

从酿酒酵母中提取pCAP01-CM导入大肠杆菌中，经PCR和酶切鉴定正确后(图7B、C)，利用PCR-Targeting[21]将卡那霉素抗性基因替换为阿普霉素抗性基因，然后将质粒进行测序，结果发现CxnD(编码404个氨基酸)中由于PCR引入了一个点突变，造成第149位氨基酸由原来的Glu(GAA)突变为终止密码子(TAA)，因此将该质粒命名为pCAP-CM(ΔcxnD)，而后通过接合转移导入S.coelicolor M1146[19]中得到异源表达菌株M1146/pCAP-CM(ΔcxnD)，同时将M1146/pSET152作为对照。

考虑到cxnD基因中引入了点突变，会由于提前终止蛋白表达而造成功能失活，因此将完整的cxnD基因导入M1146/pCAP-CM(ΔcxnD)中进行功能回补。由于pCAP-CM(ΔcxnD)是整合在基因组的φC31整合位点上，因此将cxnD的编码区克隆至含有φBT1整合酶及其整合位点的pIJ10500[22]载体上，同时在cxnD的上游引入组成型强启动子ermE*p即得到重组质粒pIJ-CxnD，测序正确后通过接合转移导入M1146/pCAP-CM(ΔcxnD)中获得含有完整的CM生物合成基因簇的异源表达菌株M1146/pCAP-CM，同时也将pIJ10500导入M1146/pCAP-CM(ΔcxnD)中获得对照菌株M1146/pCAP-CM(ΔcxnD)/pIJ10500。

质粒pCAP-CM(ΔcxnD)中仅在cxn基因簇cxnA～F中存在4个EcoRI酶切位点，用EcoRI酶切后的大片段自连产生的质粒大小约23kb，删除了大部分的生物合成基因(包括cxnABCDEF)，约5.1kb，将此质粒命名为pCAP-CM(ΔcxnA～F)。经PCR和酶切验证正确后将其通过接合转移导入S.coelicolor M1146中得到阻断菌株M1146/pCAP-CM(ΔcxnA～F)。所有重组菌株均提取基因组，而后经PCR鉴定正确(图8)。

将所有重组菌株M1146/pCAP-CM、M1146/pCAP-CM(ΔcxnA～F)和M1146/pCAP-CM(ΔcxnD)、对照菌株M1146/pSET152和野生型菌株在ISP2培养基中同步地进行固体发酵，经乙酸乙酯萃取得到发酵产物，然后进行LC-MS分析。

发酵结果分析，

一、CM生物合成基因簇功能分析

与对照菌株M1146/pSET152相比，M1146/pCAP-CM的HPLC峰形图上存在明显差异(图9A-II、III)，在18.1min和16.9min处都出现了差异峰(1、2)，提取分子离子图发现[M-H]^-分别为232和218(图9B)，与野生型菌株CPCC 200056中的CM与DCM的出峰时间和分子量相同(图9A-I)，并且两者的UV最大吸收峰为230nm和300nm(图9C)。而在阻断菌株M1146/pCAP-CM(ΔcxnA～F)中则没有出现这两个峰(图9A-IV)。以上结果说明CM生物合成基因簇在异源宿主S.coelicolor M1146中成功表达产生CM及其类似物，确证其为CM生物合成基因簇。

二、cxnD基因功能的分析

在cxnD基因功能缺失的异源表达菌株M1146/pCAP-CM(ΔcxnD)及M1146/pCAP-CM(ΔcxnD)/pIJ10500中，没有发现产物CM和/或DCM，但是在18.8min处出现一个新峰(3)，出峰时间与CM接近，其分子离子峰m/z 234[M-H]^-，比CM(m/z 232[M-H]^-)多2Da(图9B)。而且，此新化合物的UV图谱显示其峰形与CM相似(图9C)，最大吸收峰为220nm和280nm(CM的最大吸收峰为230nm和300nm)，其中波长较大的特征峰为CM中富电子基团(如O，S等)的芳香大共轭系统π-π*跃迁引起，若失去与芳香环相连的富电子基团，该特征峰将发生蓝移(向短波方向移动)。结合化合物CM化学结构，我们推测新出现的化合物可能是在生物合成过程中CM的芳香环外C-S键没有形成的产物。这一推测得到了LC-HRMS/MS结果的确证。

为了解析新化合物的结构，我们将此差异峰进行收集浓缩，而后利用高分辨质谱进一步分析，在负离子模式MS/MS图中(图10B)，分子离子峰为m/z 234.0600，对应分子式C₁₂H₁₁NO₂S(理论值：234.0606[M-H]^-)，经过一定能量裂解之后得到了多个碎片离子，其中丰度较大的三个碎片离子分别为115.9555、141.9682、173.9387，根据CM化合物同等条件下的MS/MS结果可以推导出该化合物裂解过程(图10)，因此该化合物的结构如图所示(图10B与图1)，为CM的开环结构(secochuangxinmycin,SCM，图1)。该结果提示cxnD基因编码的CxnD蛋白参与C-S键的形成。

实施例4、创新霉素生物合成过程分析

结合上述实施例1-3的结果和生物信息学分析结果，对CM生物合成过程做如下分析(图11)：

图11硫载体蛋白的活化(A)和CM生物合成途径分析(B)。

1、JAMM金属蛋白酶家族的CxnF水解CxnE(SCP)末端的10个氨基酸残基后露出双甘氨酸基序，而后先在SCP活化蛋白MoeZ(N-端为类似于SCP活化蛋白ThiF的结构域)作用下生成腺苷酰化的SCP，再在MoeZ的C-端硫氰酸酶结构域的作用下形成硫醇化的SCP，即为硫原子的供体。

2、另一方面，前体色氨酸(L-Trp)中的氨基在氨基转移酶CxnB的作用转变为酮基形成吲哚丙酮酸，然后在C-甲基转移酶CxnA的催化下形成3位甲基化的吲哚丙酮酸(3-甲基吲哚丙酮酸)，再在还原酶CxnC的作用下将酮基还原为羟基形成3-吲哚-2-羟基丁酸，而后在噻唑合成酶ThiG的作用下将硫醇化SCP中的巯基替换羟基得到3-吲哚-2-巯基丁酸，即开环CM(SCM)，最后在P450氧化还原酶CxnD的作用下形成C-S键，得到创新霉素。

最后需要说明的是，以上实施例仅用作帮助本领域技术人员理解本发明的实质，并不用做对本发明保护范围的限定。

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SEQUENCE LISTING

<110> 中国医学科学院医药生物技术研究所

<120> 一组生物合成创新霉素或开环创新霉素的基因簇

<160> 39

<170> PatentIn version 3.3

<210> 1

<211> 21665

<212> DNA

<213> Actinoplanes sp.

<400> 1

tcagcttgtc gtggcggtgg cgccggcggt tccgccgctg accagcttgg cccaggtggc 60

ggggccggcg atgccgtcgg aggtcaggcc cttggccttc tggaaggcgg tgagcttgct 120

ggtggtggcc gggccgaaga cgccgtcggc ggtgacgtcg tagccgttgt cggcgagctg 180

gcgctggagg gcggtgacgt cggtgccctt cgagcccgac ttcaccgtgg cgatcagctt 240

cgcccaggtg gcgggaccga ccatgccgtc ggcggcaagg ccctcggcct tctggaacgc 300

ctggaccttc gcggctgtcc ccgtaccgaa cacgccgtca gcagtcgtcg cgtacccgtg 360

cgcaccgagc agcaactgca cggtcgccac gtcgacgccc ttgtcgcccg ccttcaccgt 420

cggccacgac gtgcccggct gcggctgcgg cgcaccgcct cccttggcga gggcccgcag 480

ctggtccagg ctgccccggt acacgttgcg gtcgcccttg cccgcggcgc cgttcttgcc 540

ggggaggcct gggatcgcct cggactccgt gtactgccag agcgaccagg cacccgcgcc 600

cggcacgtcc tgcggctcct tcgacccgct ttcgtagcgg gccagccaca gcgggtggtc 660

cttgaagacc tggcccttgc cggccatgca gccgttcacg aacgacgccc gtgtgtagac 720

gatcggcgtc accttgaacg cctcctccac gcggttcagg aaggcggtga gctggtcggc 780

gcggagcgcc ttcgggcaca cctccttgcc gttcacccac gtgccctcga cgtccagaac 840

cggcggcagc tccccggccc tcttgccggt gtagccggcg gaccgggccg cgcggatgaa 900

gtggtcggcc tgcgcgccgc cgtccgtggt gctcttcggg tcgaagaagt ggtacggggc 960

gcgcagcagc gatgtgccgg acgcgtcctt gaagtcccgt gcgaaccaag ggtccttgta 1020

accggtgccc tgcgtcgcct tgaggaacgc gaaggagttg gactgggcga cgcgcttcca 1080

gtcgatgggc ttgccggtcg cgtcgtggtt gtggtggctg gtgtcgacgc ccttgacctc 1140

gtacgtactg ggaggggcgg ccacgctctc gtccgcggtg gacagcagga caccactcat 1200

gagcgacgcg gtggcgacgg ccgccgcggc caggcgcagt ccgtgacgac ggccgcgcgg 1260

gttctccatg gacacagtca ggtccccttt ctgaggcatg tgggcgcaaa cccgcgcacg 1320

ccgcagcggg catgacggac gggttcaagt ggtggtcgaa cgaccggaat tgggggtggt 1380

tgccgtaacc atgcaccggc gtcaccagcg agtcctacac tttcggctgg ccggctgatt 1440

catgcacaag gcccgggtga tgcagggcgg ttgagctgcc gtcatgcacg gtgaacttgg 1500

ggggacgcat aaatgggcgt tgaacgcgac gagccgacgc ggtcagcgag gcgggaactc 1560

gccctgctgt tgcggagctg gtgggaggcg caccccgaca agatcacgca ggaggcgctg 1620

gcccggcgga tcacggagcg gggcgtacgg atcagccagg agatgctgtc gcgctacctg 1680

aaccggtccc gcccgaccac ggcccggccc gacgtgatcc gcaccatgca cgaggtgctg 1740

cgccgggcgc cggaagagct ggacgtggcc ctggaactgc acgctcgggc caccgccccg 1800

cagacgccgc ccgccgaggg ggcggccacg agccagccgg ccggggacgc ggggaccgcc 1860

gcgccgaagg gcgtagagcc gacctcggcg gccccgctct tgacccgcac gccccacacg 1920

ccccgccccg cttcgcggaa gaagtggccg tggatcgccg tcgtcgcggc cgcggtcgtc 1980

ggcgcgtccg ggctcaccgc cttcatgaca ctgggcgacc agcggcagaa caccccgcgg 2040

ggacacggag cgacaccctc cgcctcaccc accgccctgg tgtcacccac cgcccagggg 2100

tcgcccgccg gcacgcatcc tcccgcggag tgccgcgacg agtcctgctt cggcatcgac 2160

gccaagtacg ccatctgcca ggacgacgcc gccacttact acacgggccg cgcccacggc 2220

gtcctcgtcg agctgcggtt cagccccgcc tgccaggcgg cttgggccaa gatgagcggc 2280

acctcgcagg gcgatgtcgt acgcgtcacc aacaacgcgg gccgcagccg ccactacacc 2340

cagcagtggg gccgcgacgc ccacaccacg atggtggagg ccgtgagccc cgacgacgcc 2400

aaggcttgcg cccgcacccc gcgcggcgag gtgtgcgcca cgaaggccgt cgcgtccgcc 2460

ccgcgcgacg cggcacctgg cgagcgcgcg gcacctggcg ggcgctgacg gccccggatt 2520

cctggccgcc ggccggacgg tgtcgcccca gggcccgggg acgccctaca taaccgcagt 2580

tcagcggcgt gcatagcggg aagctcatgc atgcatcaat gcaccgccgt agcgctggat 2640

cacgtcgcct ccagccgtgc agagtagagc cgcggccgac gagcccgctc tctccctcct 2700

gggatcgagg tgcgggtgga tccgcgatcc cgagcgtgcc gacgtacgcc ggtaccggcc 2760

gccgcccctg cgccacacgc ggacgcaggg cgcagaccac tgccacccaa ggccaaggag 2820

tcagccatgc ccgcacgcac cacacgaacc gcacacacca cacgcaccgg ccggttggcc 2880

gtcgtcgccc tcgcggcctt gacctgtgcg ggcctggtca ccggaactgc agccacggcc 2940

accacacccg actccctgcc caccgcgaag cgcgccgcag cgcccgacgc agcggctgta 3000

tcgtggccga cgctgaaggc gggcgcgcgc ggtacggagg tgaccgcgct ccagcacctg 3060

ctgatcgccc gcggccaatc cgtcgccgtg gacggggagt tcggcccggc caccaccacg 3120

gccgtcaagg cgttccagaa ggccgacggg ctcaccgccg acggcatcgt cggacccgcc 3180

acctgggcca agctcgtccc gacgctgcgt cagggcgcgc agggcgcggc ggtgaaggcg 3240

gcccagaccc tgctgaagac ccgtggccaa tccgtcgccg tggacgggga gttcggttcg 3300

gccaccacct cagccgtcaa ggcgttccag aaggccaagg ggctcagcgc cgacggtgtt 3360

gtcggcacgc agagctggtc cgcgctcctc acctcggact ccggcgcgcc gtccgggaac 3420

cgggccgcgt tcgcccagca gatcctcaac accagcggca tcgagctggc gaccgtccac 3480

cccggcggca cccacgccgg ctccaccgcc cggcagaaca tcatcgacac agccaacggc 3540

aagggcgctc tgaccagtcc ctggagcgac aagccgaacc agcgcgtggc gctcgacacc 3600

cggatgctca acgggctgct gaagctgctc tcccaggacg gctaccggat ctctgtctcc 3660

gagatcgtcg gcggcgacca cagcacgaac tcccggcact acgcgggact cggcttcgac 3720

atcaactaca tcaacggccg gcacgtcggc gagagcgccc cgcaccaggg cttgatggcc 3780

gcgtgccgga agctcggggc caccgaggtg ctcggtccgg gcgacgccgg ccacagccgc 3840

cacgtccact gcggctggcc gcgctgatcc cggctgaccg ccaacttccc gtgcctgcaa 3900

gcagagaggg tccgtcagga agcatgacgg accccctcat gcaggatcga gggtgacgtc 3960

cgggctactc cgtagcaccg tccgatttct tgccggctgc ggccaaggtt cctcccaata 4020

gggccgcgcc caggggccat gaacgcatgt gcacggggag ggtcacgcag tgatccccga 4080

cgcgcatcac aaggcccttg ttcaccaaat cgcgtgcggc ggcggcgaat tgcgagtttc 4140

gggtcacctc ggcccacgcg catccctcca ggcccaggag gaagacggcc atctgggtgg 4200

gatcgtccac gatgatttcg cggctggatt cggggcgctg atcgacgacg gacaggaact 4260

tcgggccctt acggaagtag aggaggccga aattgctgga ggaccgccac tcaccgacgg 4320

acggatgccc tgtctccagc accgtgatgc tgtcgggggc aggaaggtga tcgagacggg 4380

ggaccaggtc gagctgttcg gcgcccaggg tcagtgacca ggtgactctg gtgccgatcg 4440

aggaacattc gcgaatcaac gcgatgatcc gcacgatcac gtgactgggg agtgccgaga 4500

agtcggcggg ctccggcagc cgtaccccgg cgacgggcac ccggtgcagt agcgccgcca 4560

gttcggtggc gacgggaagt tcggcccggt cgagtccgag ggtcagcgtc tcggtcgcgg 4620

gacggccacc ggccggtacc cgtccctcgt ccgaatctcg ggccttgagt tcatcgatgt 4680

ccagcaaagc gctcatagcg aagccgccac ctccttgccg ccgaccagct ttcggcgata 4740

cgggtcaacc ccgagggcga cgcttacgta gcgcccctcg tcctcgaatg ccaggccgcg 4800

atcgacgaag tagcggagca tttcctcgag ttccgcttcc ccgacgacgt gcccgctgtc 4860

ggcaagccgc cggcgtatgc cctcgcgggc ggcgcactgg aacatgccga ggtacacatt 4920

gctgcggacc tcgtccagct cgatcacttc cgtcggccag ctggcacggc ggtcttcgat 4980

gacgacccgg cctcggtcat cggtccagta ggaaagggtg ccctgcggat aggccttggc 5040

ccattcctcg caggcctgct tcatctcgtc ctcgatgggc cctgagattc cccggacgct 5100

ggtgtcgaag aagaacacca tgtcgtacag ctgatcctgc gggatctggt agatgaagtc 5160

gtatatttcc gaggggcggc ggaacatgaa cccctgggtg gggtcctcga agtagggact 5220

gaaccgctca agggctatgc gccaagcccc ggttggcggc tccaggtgct cgagcgtggc 5280

caatttcttg agcagcccgc ggtagtcgtc ctcggtctcg cccgggaagc cgtagaggat 5340

gctccatgtc acgttgagcc cgagatcctg tccgtcacgc agcatccgta cgttgtgcgc 5400

ggcactgacg cccttgtcca tgaggcgcag cacatggctg ctcaggctct cgataccggg 5460

ctgcacgaag aggacgttcg cctctttcag cctactcaac tgctcccggt tcatattgga 5520

cttgatctcg tagtgaattc gcagatcgca gtcgagggca gctatctcgg gcatggccgt 5580

attgagatac ttcatgtcga ggatgttgtc caccatgacc aggtcgagga tctggtgtcg 5640

ctcggccagt tcccggactt cctgggcgat gcgctcaggg gccttgctcc ggaagtcgat 5700

attcgatccg ttcaggccgc agaacgtgca ttggtgagcc tctccccacc agcaaccacg 5760

ggaggtctca aggaccagca tcggacggac gtggtgacgg acgggtgacc tttcgagggc 5820

ctgaaagtag ctgtcgtaac cgggcgcggg caccatggcg aacggcagcg ccgccgtggc 5880

cggtggattc accaccggat gcccgtcatc ccccctccag ctgagccccg gcacgtcggc 5940

gaggctctcg ccccggatga tgcgattcag caacgcgggc agcgcacgtt cgccctcacc 6000

gctgatcacg aagtcgagtt gctcgaaatt ccggtgcaac gcgggacctt gtgctccgtc 6060

gcagttgctg ccgccaagga ccgtgcggat gcccggcgcg agtttcttca gctccctggc 6120

cagtgcgagc gacgggacgt tctgcatgaa ggtgctcgtg aacccgacca cgtcgggagg 6180

atcggcagcg atctcggccg cgagatcccg gatgaatccc cgggcgtact tgtgcatctc 6240

aacgggaagt gtcgggtcca tgtcccgctg ctcgaggaac ttcgcgtact cgtcgacctg 6300

ataactgtcg acgtcgtaca gcgctggggt gaacacccag tcccctacgc cgtggaagac 6360

ttgatccgcg atgttcccgt agtcctcgca ggtgacggag ccgttgctct cccgcatcag 6420

gtattcggcc cagcggaggt tggcgtacag ctcatcgacg gtccagtcgg cggcgttctt 6480

gcggacgcat ggccccagta cgcccagcgc gctggacggc gtgtcgagcc cttgccacgg 6540

catggcgatc atcaggagtt tcacagactt cccaccccag agttgctgtc gatgagttgt 6600

atgaggacgg gcaaaagaat tgtccgcaga gagttctcag aaccagttcc cacggtccgc 6660

ccggccgagc gccgccaaca gcgtccgatg tgcctcggcc tctcccactg agatccgtac 6720

accgtgcccc ggaaaggctc ggaccttgac ccctgcggta gccgcagtcc gggcgaaaga 6780

ctcggcggcc gaagcgagcg ggagccagac gaagttggct cgggaaagca ggacgggcag 6840

cctcagttcc ctgagttccg cggtcagttc ttcgcgtgcc gcagccactg ctgccagacg 6900

ttcacacagt tcgtcctcgc tgcgcagcga gagcattgcg gcttgttccg cgaagcgcgt 6960

cactccgaaa gggattgccg tcttgcggac ggtggccatg acctgccgtg gcccggccgc 7020

gtaaccgacc cgtaggccgg caaggccata ggccttggag aacgttcgaa gtaccacggt 7080

gttgctgtgc tcgctcagca acaccggcag acccggagga ttggcgcccc ggtcgaactc 7140

cacgtacgcc tcgtcgagga ccgcgaccac atgagccggc agcgaacgca ggaaaccgtg 7200

cagctcgtct tggtcaatca cggttccggt cggattgtgc ggggagcaca ggatcaccac 7260

cctggtccgc gcattcaccc gggtgcggat ctcatcgaga tcgtggccgc cggacgcagt 7320

caggggcacg tggactccgg tggcacctga aatggcgacc aacagcggat aggcatcgaa 7380

tcccggccag ccatggacga cttcgtcgcc cttgccgcac agtgcgagaa ggatctgctg 7440

gagcacgccc gcgcttccgg ggccgaccgc gacctcatcc ggggagacgc acaagtgccc 7500

ggcaatgtcc tcggtcaggt cccgtgctgt ggggtcgggg taacgagcaa gtcgcggcaa 7560

gcctttttcg ataccggcaa gcacggtagg cagcgggggg agaaccagct cgttgctgga 7620

caggtcgaag gtgaaccgcg agctgccttc ggcgttcgac gactccttgt cccggtaggc 7680

ccgcatgtcc cgcagggtgc tgcgttctgc gaatctcacg ttcacgcctg cagactcctc 7740

gccgtgcgtg attcgcgggt ggcgagcagc tcgtagagca tttcgtggac cggcgcggtc 7800

aggcccgccc cgcgggcgag gcggaccacg gccccggtcc acgcttcgag ctctgacggc 7860

cgtcccgcca ggatgtcccg ttgcagcgag gaggtgacgt cgggcgactg ctggtccatg 7920

agctctgtcg cggtgtccac ggcagctgcc ggcagcgcga ttccgagctt gatcccggtc 7980

tcgtagatct cccgcatgcc ggcgatcaga atgttgcggg tgccggtgcg cgaccggagc 8040

tcgccgatgg tcgccccgcc ggtggcggct ccaaggctgc cgatcgggac caccaacagg 8100

aacttcgccc aaaggccggc ccagatgtcg ctcggctcgg gcacggacac cgaggcagca 8160

cgcagcacct cgcgcagtcg tgccacccgg tcggacacag tgctgtccca ctcggtgaag 8220

gccagagcgc cggggggacc cacgtgcctc aactcgcccg gaccggccgt cgaggccacg 8280

accctgacgc tgccggggag tacccgaccg cggccgatcc tggctgcgac ctgctcaggg 8340

gcttccaccc cgttctgcac cgtgaccacg gcagtgtgct cgccgaccag cgggcccagc 8400

gcgtcgaggg ccgccggcag ctgtgaggtc ttgacgcaga gcagtacgaa gtcgacctcg 8460

ccgatgtcct tcgggtcggc cgacgcccga acgtccggca cacgtaagtc acttgagccg 8520

ttggtgatgc gcagcccctg tcgcctgagc gcggcgaggt tctcgccgcg ggccaggaac 8580

cgcacatcat gcccggcggc ggcgagcagg ccgccgaaat agccgcctac tccgccggct 8640

cccaccacag caatgctcgg accgccttgt tccgtcatcg cggcaacctc tctcccactc 8700

gtatcgggag ggcctcgagg ccccggctga gcgaactgtc ggagagccag gcgacctctt 8760

ccggaggcac ggcgagctcg aatcgcggca gtcggcgcaa cagcgtggag aaggcgatct 8820

cgccctggag tcggcccagc ggagcgccga tgcagaagtg gggaccgtgg ccgaaggaca 8880

gatggcggtt cggtgaccgc gtcacgtcga aaccgtccgg gtcgtcgtac acgtcgggat 8940

cacgacccgc gctggacagc gacaggtgta cgaagctccc cttgggtatg tccactcccg 9000

cgagctgcat gtcctccgcc gcgacccgca gcgaggccca ggcggccgac ccttcgtagc 9060

gcaggatctc ctcgatcgcc gaagggatca gctcgggggt ggctcggagc atctcgagct 9120

gctggggatg gcggagcaac agtgctgtgc cgttgccgat catgttggcg accgtcttgt 9180

gaccggcgat gatcagcagg aggagcgtcg aaaggagctc tgtttcgctg tatacgcctg 9240

cgtccctggc cctgatgatc tcgctcagca ggtcgtcccg caggtccgtg cgccgctcgg 9300

ccacgagctt ggtgaaatag tccgtgaact cttcgctcgc ggctttcagc tccgcctcgt 9360

cgtgctgcaa cgggtcctgg ctgaggatgt agctccactc cagaaacagc ggccggtccg 9420

ccaccggtat ccccaggtat tcgcagatga cggtgagcgg catcggcaga gcgaaggaac 9480

tgagcaggtc gatttcgccg ttctccggaa aggtgtcgat gagatcgtcg acgatgtcct 9540

ggatgcgcgg gcgtagttgg gcgaccgtgg ccggcaggaa tgcccggctg atgggcttct 9600

tcaggcgggt gtgtttgggc gcgtcggcga atcccaggtt gccgaccacg aggagactgc 9660

tggccacggc cttgtctcga tagcgggcgg gcaggttctc gacttgcttg gagaggcgcg 9720

agtcgccgag cgcttcctca acgactttgt tcccgaggac cgcgtacgcg tcggcgccgg 9780

ggggaacatt gatccgatgg accggacact tggacctgta ctccgcggct gtcgcgtgcg 9840

gattcgaacc gggctcggtg aagaattccg tgggaatcac gtcggtcatc gtgactcctc 9900

ggctcgcgcg gcttcgctgc cacctgctac cgccggcact atccagatga cgtcgtggtg 9960

ctccactttc gtttcgaggc cgtcgaggct cctgatgtcg ctgtcgttcc ggtagacatt 10020

gacgtagcgc ttcacggacc cttcctggtc catgagtcgc tcgaggaccc ccggacaggt 10080

ctggtcgaga ccgacaagga cctcccggat attggcgccc tcgacaggca actgccgccg 10140

accgccggtc aggacgtgga aggctgcggg aagtttgaca tcgggcattt cttattcctc 10200

ctcgagtagg acttcgaact gctcatggat cgccgccgga atccgtatgt caagcctgtc 10260

gcattgctcg gcaccgggcc gccaggccgc tacccgatgt gcgataccgc cactactctc 10320

atgcacgtac cagatgacgt tcactggcca gcatgactgc cggaaaaggt attcatccat 10380

ctgtgtcgga tgttcgctca ggcgctggcg ccgttcgtgc ggcgggccga tttcatgcca 10440

gttcggatgt gaatgtatgg agcccagcaa ctccaggccg ttcgccgact gttgcctgat 10500

cgcctgcaag acgccctgtt catcggacca gaatcctcgc cccggattct tgtacacgtc 10560

gccgaactgc ggggcgatcg tcgcttcgaa ttcggccatg acactctcgt cactgtcccg 10620

gacattcggc acgaattcca catcgctgat cactatttca gcgccgccca cctgcccgaa 10680

caacagaccc gatgcacgcg gcagatggct cgggacgcca tcggcgtcgc ggctgtccag 10740

gcatttttgg tactccccca gtgcgctgct caggaaacgc ctgacgggtt cgatttcaaa 10800

tcggacggtc accggcagat cgtcgagcag ggctgcactg tctgctgtgc cacccagacc 10860

gtcatccgta gcagctgtca catactgggc atctttcacg cctgccattt acaccctccc 10920

tgagatttcc tggtgaaatc cttaggccct gggcagtcgg tggaattgcg gagcgttcat 10980

gggcgtacgc cccctgcgct ctggcgaggc aggtcagtca cccctggcat acgagggccg 11040

cggagcgtgg aggtcttgat gccgacccag cacaacagcc ctccggccgc cgctacggcg 11100

gcagcgatcc agaaggcggt ggtgaagccg tcgcgcgccg gcgtatcgga accggagata 11160

tgccaggcag ccagcaaggc gacagccagc tgactgccta cgacgccgcc tgccgtgcgc 11220

accaccgtgt tgacgccatt ggcggttgcg gtgcgtcggg cctccgtcaa gtcgctgatc 11280

accgacggga gtccgctcag caccagtccg gaaccgagac ctacgaccag gtaaccgacc 11340

gccacttgcc acccgttggc attccacgcc cagagcgaga tcgccccgac cgccatcacg 11400

gcgaagcctg atgcgagcgt cgcgcgtacg gacgtcaggc gctgcagcag acctgctagt 11460

ggtcccgcag gcaacagcac caatgagccg ggcagaagga gcagacccgc catggtgaca 11520

tcggccccga gcccgtagcc gatggtccct ccgcccggta gccgctgatc cgccgcggtc 11580

tgggcgtacg tcggcagcag gacgtagaac acgaacgaga ccacgccgaa cacgaacgcc 11640

gcaccgtgca ccgagacgaa ggagcgaccg gccacgaccg cggggtcgat caagggggcg 11700

ggcgactttc gttcgactac gaccagcaat cccaggagaa ccgcggaggc cccgaacaga 11760

gccagcgttc ctgtggacgc ccacccccat gaggtgccct tggtcaacgc gagcaacagc 11820

gcgacgagga ccaaggccag gagcaccgcc ccgggcacgt ccaccggttc gccggcttcg 11880

ccccgctggt ccggaacgta cttcgcaacg agcccgatcg cgcccaggat cagtaccgcg 11940

gcgacggcga acagccagcg ccatgactgg tggtcgacga ccagacctcc cacgaccagg 12000

ccgatgccgg cccccacacc gatggtccct gacaccagcc cgaggcccga acgcagccgc 12060

tgctcaggaa gtacgtcgcg caggatgccg aaggacaggg ggatggccgc gaggctgact 12120

ccctgcacgg cacggcatgc gatcagaacc ccgatattgc ctgccacagc acaaccgacc 12180

gtgccgatca ggtaggtggt gagaaccagc agcaaaactt tgcgcttgct gtagcggtcc 12240

cccagccggc tcagcaaggg cgtgctcgcc gcactggtca gcaggaacac actcaggatc 12300

cacgccgacc acgtcgaggc cgtgtgtaac tgcacctgta gtacgtgcaa ggccgggacg 12360

accatggtct gcatgagtgc gtacgacagc acggagacgc cggtggcgat cagcgtcatc 12420

gtcgcgctga tcgacggtgg acgtggcgaa gtgccgttgt aggacaaggg ggacccccgt 12480

ggggatgtgg tcggggcggt tactgagtca cgagaccact ggccggagcc acatcactga 12540

ggcttaaaca ctgcctgcgc aaagaatccg tcaattgtgt ttggggcgat acacccgctg 12600

aaagcgactt cgtacggcaa agacaagaag acctccggtg gaattttcca caggccccgc 12660

agtcaaggct gagattcgat agctgcacct tgcatgggtg atcgtatgta gccgcctaca 12720

tacacgccaa tacccgttgc gtatggggcc atatactgat catatggaac tggatttgcg 12780

gcacctcagg tacttcgttg ccgtagccga ggaaggcggg ttcacgcgag ccgcggcccg 12840

cctgcacatg acacagccgc cgttgagcgt ggcgattcgt caactcgaaa gagagctggg 12900

tctccagctt ttggacagaa cgggcaacag agtcgaactc acgtcggtcg ggcgcgactt 12960

cctgactcac gcgaggaact tgttgcagca gtggcaggtc acggtcgaga ggatgcggca 13020

ggcggggtcg caggatgtcg aacggctcgt cgtcgcgttc cgcccggccg tcagccgccc 13080

tctggcacac cggaccattg aactcatccg cgaaaagcac cctgagtatc aggtagtgcc 13140

ccggtacgta ccgtggaccg aacagacagc atgcctggag gcaggggacg ctgacgtgtc 13200

cttcgtgctg gagcccgcgg actacgtggg cctcgagagg gccaccgtgg ccctgttacc 13260

ccgggtcgtc tgtctgccat cggctcacga gctggccagt cgtgactccg tgtcgatcga 13320

cgacctgagc gaggttccga tcattcgccc caccggcggg tcgcccgagt ggtccgactt 13380

ctggggtggt gaggtgtgcc ccggcaagcg cacctggaag gaacctccca cagcgacgcg 13440

cctcgacgag gccatcgacc tcgtggccct cgagaacgca gccgcgctcg tccccgtctc 13500

tgtcatggca gtccagcacc gtcaggacgt cgtcttcatc cctgtgacgg atgtgcctgc 13560

cgcccggttg tcccttgcct ggcgtgaggg ttccgactcc gaactggtac gcctcgccgt 13620

caggtgcgct caggccgcag cccaggatcc agccgtcagg acgctcttcg gagaacctcg 13680

accaaccgga accgctccgg cctgatgaga gcaggggctg tcaggaactt cctgatggcc 13740

cctgccagtt gtggtacgtt cgcgatcatg ttgcgcagga atctttgttg gtgggccgcc 13800

tagggcggcc caagaacctg cgctcgaccg agccagccag ccgctccgag ggacgggctg 13860

ctgaacttgg tcggcccgcc tcgtcgatgc ggtcaagacg agaggatcag ggccaatgtt 13920

cagtctttcc gggatcacgc cttcaggtaa ggcgcacctg ggtaactacc ttggggcagt 13980

gcgtcgttgg gcagcacagt cgggccccga agacctgtat ttcgtcagca acctgcacgc 14040

catgacgacc aagcacgacc ccgaacgtct ccaggaactg accgaccacc aactcgcttt 14100

actcatcgcg gcgggcgtac cccaggaacg tctcttcgtg cagtcggacc tcatccagga 14160

gcacatggcg ttgacgtggc ttctcgagtg cacctgcacc ttcggggagg ctcgaaggat 14220

ggtgcagttc aaggagaagt cccaggggag caactccgta cgccttggtc tgctcaccta 14280

ccccgtcctc atggcggcgg acatcctgct tcatggcgct tcagaggtgc ccgtcggtca 14340

cgatcagaac cagcatgtgg agctggcccg gaccttggcg cggcggttca acacggacta 14400

cggcgaggtg ttcacggttc cgcaagccgt cctgcccgta gccgcagccc gggtacgtga 14460

tctcgctgcc cctacgcgga agatgtcgaa gtcgtcctcg gacggcagcg gcatcgtcta 14520

cgtcctggac agcccggagg ccgtacgccg gaagttccaa cgcgcagtga cagacggaga 14580

aaacaccgtc cgctacgccc cggacgaaca gccgggcgtt gccaacctcc tggagatcag 14640

ggctgcctgc actgacacgc tcccgagcga tgcggcgaag ggtatcgatt cctaccgtga 14700

cctcaaggaa gcagccgcag aggcagtgat ctccctgatc gcaccggtgc gtgagcgggc 14760

actgcagctc ctcgaagagc gatcggagct ggcgaagatc cgggctgagg gggccgaccg 14820

tgctcgggcg cggtcacgag accgcttgga tcgtgcgctc agccttgccg gtctgaagta 14880

gcagatcacc ggccgggctc tggctcacag gagctccgga gtccggccgg gctctcgatc 14940

aagtgatgcg cccgagggtc cctcatgcgt aaagatgtgg cgcaacggat gccagtgggg 15000

ggagcagatc tgttgcgtac gcgcgggatt gccggggtcg tgtcggcggt gctgggcgtt 15060

cttctcgcga tatcactcgc aactgccccc gcccatgcgg cagttcgctc ggccgcggcg 15120

gtcgatgtct gtcggtcggc cgccctgagc aaggcgcgtg tgagcacgtg ggtgcggctt 15180

gagcaccgcg atggtacgta cagcaggatc cgcagcgagc tcagcgtcga ggtgcccgag 15240

gattggccgt tggccaagga cctgctgctg agtgaggaca gccgccggta cgtcgcggcg 15300

atgtcctgcc tcacccgtac cgatcggggc cggcaacgcc gctggtcgga gtggaggagc 15360

agccgtccga cggtggcgtc cacgaagagc ggtggggtga aggtcgtcga ccgtacgcac 15420

tcctgggtca acgtgtatcg ggcgcacatc gatgtgggta cctggcgggt ccgtgcgggt 15480

gcggagcgct ggaccgtaca actgcaagct ccgtccgcgc tgaacgcggc ccgctgggat 15540

gagatcaggg tggaacccgg cgccccggga gccgagtcgg cgaccccgcg gcctgacgag 15600

gggcgcggcg ccacggcgtt ggtgtggcat ccccagaacc accgtgagaa ggcggctgct 15660

cctgccgtga gcgttgcgct caagccctcc tggcagcgtt cgtgggcagc ccagaacgac 15720

cggctggtcg ccgtggcgct ggatcggggc ggatggctgc tctgggacgc gacgagtgcc 15780

gccctgttgc tgtacgcaac cgtcctgtac cggaggcgtt ccgctcctcc cactcaggct 15840

caggagcgca cactgcgcaa tctttccctg tgggccaagg ccctcgtggt gctggtcgcg 15900

ctgacgagca tggacgacgt gctcattcgg tacgtgcaac ggcggggcga cgggctgttg 15960

ctggacgagc agatcccgcg cgggaatgcg ttcgccctgg cagccgtcat cgtgctgttc 16020

tgcgtcggca ggccgcgtcg gcggatctgg gcggcggctg ctgtgctggc cgtgccgacg 16080

gtggctgcct tgccgcagtg gttcgaactc tccccgcagc gcttcgtgtc cgacgacgag 16140

tgggcagtca cgttggcggc ccagggggtc gccgcctgct gcatgctggc tctcttgggg 16200

ctcggcttcg taactgccgc ctggcgcttg gccgttgacg gggacctgct gccgatgagc 16260

cgtcggcacc cggggcacgc ccgggtcctc aggctccgca tcgccgggcc ggtgatcctg 16320

gtgtgtacgg ccgctgtggc gatctgtttc gccctggccc aggagcgcaa ctggcagcgt 16380

gccacctggc tcagcgatcg ctcggacccc gcctacgcga ccggccagtg gagcgatcgc 16440

gtgtgggagg cggtgtggtc cgtcgccaat gggcaggact ggctctcgtg gcaggcctgg 16500

ctgctcacgg gagttgcggt gcttgcggtc ttgcgcacct ggcgcgcccc ggcctccgtc 16560

tcccctctgg acgacccggc ggaccgcctt ctgttcctcg ccttcttcgc catcgtggcc 16620

gcggcttccg gcggctactt tctgggcaac gaggtgctca ccggcttgtg gattccgctc 16680

agcatgctgg ctctctactg ggtggtggtt cccttcaccc accgctcggt actggcgcag 16740

cctttcgagc ggtccgggcg gcccctcgcc gattccgcgg ggcccggcgc acgcaccgta 16800

ctgcttgcca aggcccgctc ctaccgcgag acccatgccg aactgcgccg cctcgaccag 16860

gggttgttcg gggacgtgcc accgaagcga agcgacctgg aacaggagtt gagcgacctg 16920

cacaactggc ccacggcagg tggctccgac cggcttcccg ccaaggtgtc cgtggtggac 16980

ggagcactgg cgctggggcc acgagacacc tggtgggcca atggcagccg ctgtgcccgc 17040

ctcgccttgg ttccggcggt accggcggcc ctgctcctgg cctgggtctg gaaggtcaag 17100

ggcgaggcct ggcacgcgac tctgcacgaa cagttcggtc tgccggatgt cctgctcttg 17160

ttcgtcgggg agatggtgat gttcaccagc tcggcgttcg tcctgggcgc gctgtggcgc 17220

catctgccag ggcagcgcgg cgccgccaag gccctgccgg tgacactcgc cttcgcgctg 17280

cctatcggct tggacgcgct cgtctaccgg ttcaccggcg agagcaccgc gaacctcgct 17340

ctggctgtgt cggcgatgct gttcgtgctg actgtcacca gcatcgctct cgacttcgac 17400

acgttccgcg gcgaacggcg ttactggcag agccggttgg gcctgctcct ttcgatctat 17460

cagatgcgtt actactcgct gcaggccgcc tacctgatcg cccaggtcgt tgccatgatc 17520

acgatctggg agttcttcgc ggaacccgac gtggtgccga agccctccga ctcgaagtga 17580

gccgggcgca ccctcccgta ggttacgggc gccactggtc ctggctttgc ggtagtcctt 17640

ggtaggtgcc gtagtccgtt gagccttggt gggtgccgta gtccgttgag ccttgctcca 17700

tcgtggggtg ctccgtcgtg gagtgctccg gcgtggggag ctccgtcgta gcgtgctcca 17760

tcgagccgat ccaggcgcgc gccggggggc gtccgacgta cttgccgaag agcagtgccg 17820

cggcggtggc agcgagtacg ccggggacca tctcgtagac gcccgattcc agcggcccga 17880

gaagcgggtc gatgtacttc cacaggaaca cggtgagcgc acccgtcacc atgccggcca 17940

tcgccccggc tgccgtcatg cgcggccaga acagcgacag gatgatcacc gggccgaagg 18000

ccgcaccgaa tccggcccag gcgtacgcga cgatgtcgag cacggcgccg ccgctcagcg 18060

cgatcgcata ggcgaccaat gccacggcca ccacgctcag tcgtccgacc atcagcagca 18120

acgtgtcgga ggcccgccgg ttgaggaacg cccggtagaa gtcctcggtg agggacgtgg 18180

ccgagaccag cagctggctg tccaccgtgg acttgatcgc ggccagcacg gccaccagca 18240

ggattcccgc gatccagggg ttgaccaggt gtgtggacag ctcgatgtag acggtctccg 18300

ggttgtccag cggctcgtcg agcacggcga tccccgcaag cccgatgagc gaggaacccc 18360

ccagtacgac gaccacccag cccacaccca gacggcgggc cagcggtatg tcctttgtgc 18420

tgcggatacc catgaagcgg atcaggatgt ggggttggcc gaagtagccg agcccccagg 18480

ccaacagcga gatcatcgcg atggcgccga gcggctcgcc ggccgaccac gtgttgccgg 18540

cgaaggatgc ctcggccacc gggtcgagta gtgccggggt cttgtcgctg agcgcgtcgt 18600

gcagcgcgcc gaagccgccg agccgccaga gaccgagcgc ggggaggacg agtgccgcga 18660

ggaacatcag cgtgccctgg atggagtgcg tgatgctcac ggcccggaag ccgccgagga 18720

tggtgtaggc aacgatcacc acggcaaata cggtgagccc gaactcgaag tcggcgccga 18780

atatctcgtt gaacaggaga ccgccggcga ccagcccgct ggcgacgtag acggtgaaga 18840

acaggaccgt gacgatggcc gagagcagcc ggagcatcct gctccgatcc tcgaaacgtt 18900

cttccaggta cgacggcagg gtcacggagt tgccggccag ctcggtgtag gtgcgcaagc 18960

gaggtgcgac aaaccgccag ttgagatagg tgccgacgat caggccgacg gcgatccagg 19020

tggcgccgat cccggccatg tacacggcgc cgggcagacc cagaaacaac cagccggaca 19080

tgtcgctggc gccggcagac agggcggcca tcggggcggt gagtcggcgg ccgccgaccg 19140

tgaagtccgc gaatgtggcc gtttcctttt gcgtcatgac accgatcatg accatcgcaa 19200

tcagaaagac cccgaaagtg atcatggctg ggacggtcag ggtgagcatg cattactccc 19260

tgcaatgcgc ggacgcgacc acctgcatgt caagtacaca tacaggccat cctcttcggg 19320

ctgaagaggc ggagtgtagg ggcctcggcg cagaccggcg agagggtctc ctgccgcctg 19380

cgtacatcgg ttccggattg tgatccctga tgtttaccgc agatcgtgta catgccaccc 19440

caggtgggta actacccgac cgcctccccc catgggcctt gagcgatgcc cggtgaagcg 19500

gctgccccgg catgcccttc ggtggtggcg ctcctgttga caggattcgt atgggcagtg 19560

cacttcggct gggcagtgca gttcgacgat ctcgtagacg tatggccgtt gcctgtggcg 19620

tggtcatcgc cgtggctggt gggctcctgt gccccgttgt cgccgcgccg accgccggtg 19680

cggcggacca tgactttcgg ccgcagttgg tgaccgtgga cacccccacc cgtgccgcca 19740

aggagaaact tgccgggctc gggctcgacc tgaccgagca tgccgggcat ggctttgtcg 19800

aagtcgtgct gcacagcccg gccgacgcgc tcgcgctgca agtgggcgga ttcagctgga 19860

aggttcgcgt acccgatctc gtccagcgtg agtccgacgt gaacgccgcg aaccgggcct 19920

atgccgccgc caccggcacc tcgccgctgc cgtccgggcg ggacagctac cgccggctcg 19980

ccgactacaa cgacgatctc ggccggatgg ccgaccagaa tcccggactc gtacggaagt 20040

tcacgctcaa gcacaagagc ctcgaaggca agcccgtgca cggggtggag atcacgcacg 20100

acgtcacggc tgtcgacgac gggcggcccg tcttcctgat gatgggcctg caccacgccc 20160

gcgaatggcc ctccggcgag cacgccatcg agttcgctca tgatctcgtc aggaactacg 20220

ggagcgatga gcggatcacc tcgctgctcc agaaggcgcg ggtgctcgtc gtgcccgtcg 20280

tcaacgtcga cggctttgaa aagtccgtca acgatgggca gttgatcgat ctgcgggaga 20340

tcgacgacgg cggcaccgga tcgatcctcg ccacgcccgg caacgcctac aagcgcaaga 20400

actgccggat cgtcgacggc ctgagcccgg tcgcgggcga gtgcgcgctg gcgagcagcc 20460

ccggcgggtt cggtgccggt gtcgatctca accgcaacta cggcggattc tggggcggtc 20520

ccggcgcggc cgccgagtcc gtgcaggcca cgtaccgcgg cgccgcgccg ttctccgaac 20580

cggagacgca gaacatccgc gagctggtca gcagccgcca ggtgaccggc ctgatcacca 20640

accacacctt ctccaacctg gtgttgcggc cgaacggggt cgcgcccgac acggtcggtc 20700

cagacgggca gcccatcggc aacccgccgg acgaggccgc actgaaggag ctcggcgacc 20760

ggatggccga gcagaacggc tatacgagtc aacacagttg ggagctgtac gacaccacgg 20820

gcaccaccga ggactggtcg tacaacgcga cgggcggcta cggatacacc ttcgagatcg 20880

ggccccacga gttccatccg ccgttcccgg aggtcgtcga cgagtacgtg ggcgcgggcg 20940

agtacgccgg gaagggcaac cgtgaggctt tcctgctcgc cctcgagagt gccgtcgatc 21000

ccgagtcgca ctccgtgatc agtggcaagg ctcctgccgg ggccacgctg cggctgaaga 21060

agacgttcgc cacgcccacc tggtcgggca cgatcaagga caccctcgac accacgatga 21120

ccgtcggcag cggcggcagc tacacctggc acgtgaaccc gtcgacccgg ccggtcgtca 21180

aggcccgcca gatcgaggtc atcggctccg agccgctgaa gcggcagacc tacacgggca 21240

cgaccgcgcc cggacagccg acggagcagg agttcgtcgt cgaccgggac gccgacgtct 21300

tcgaagcgaa gctcgactgg gccacgcccg acgacctcga cctgtacgtc ctgcgcaaga 21360

acgccgacgg cagcctcacc caggtcggca gttccgccgg ttccgtcggc gagaaggagc 21420

gggtcctcct cgacgacccg gagcagggta cgtacgtact ccgcgtggag aactgggctt 21480

ccgtcgcccc cagttggacc ctcaccgcgt ccctctacga cgccaccgtg gacgagatcg 21540

gcggcgtcat cgagaactgg acgctctcct gcgagaagga cggaaaggtg cttcagcagg 21600

tgcccgtcgt cgtcgaccgt gggcagcggg tcaaggcgga cttgaagaac tgcgcgaagg 21660

gctga 21665

<210> 2

<211> 1872

<212> DNA

<213> Actinoplanes sp.

<400> 2

tcatagcgaa gccgccacct ccttgccgcc gaccagcttt cggcgatacg ggtcaacccc 60

gagggcgacg cttacgtagc gcccctcgtc ctcgaatgcc aggccgcgat cgacgaagta 120

gcggagcatt tcctcgagtt ccgcttcccc gacgacgtgc ccgctgtcgg caagccgccg 180

gcgtatgccc tcgcgggcgg cgcactggaa catgccgagg tacacattgc tgcggacctc 240

gtccagctcg atcacttccg tcggccagct ggcacggcgg tcttcgatga cgacccggcc 300

tcggtcatcg gtccagtagg aaagggtgcc ctgcggatag gccttggccc attcctcgca 360

ggcctgcttc atctcgtcct cgatgggccc tgagattccc cggacgctgg tgtcgaagaa 420

gaacaccatg tcgtacagct gatcctgcgg gatctggtag atgaagtcgt atatttccga 480

ggggcggcgg aacatgaacc cctgggtggg gtcctcgaag tagggactga accgctcaag 540

ggctatgcgc caagccccgg ttggcggctc caggtgctcg agcgtggcca atttcttgag 600

cagcccgcgg tagtcgtcct cggtctcgcc cgggaagccg tagaggatgc tccatgtcac 660

gttgagcccg agatcctgtc cgtcacgcag catccgtacg ttgtgcgcgg cactgacgcc 720

cttgtccatg aggcgcagca catggctgct caggctctcg ataccgggct gcacgaagag 780

gacgttcgcc tctttcagcc tactcaactg ctcccggttc atattggact tgatctcgta 840

gtgaattcgc agatcgcagt cgagggcagc tatctcgggc atggccgtat tgagatactt 900

catgtcgagg atgttgtcca ccatgaccag gtcgaggatc tggtgtcgct cggccagttc 960

ccggacttcc tgggcgatgc gctcaggggc cttgctccgg aagtcgatat tcgatccgtt 1020

caggccgcag aacgtgcatt ggtgagcctc tccccaccag caaccacggg aggtctcaag 1080

gaccagcatc ggacggacgt ggtgacggac gggtgacctt tcgagggcct gaaagtagct 1140

gtcgtaaccg ggcgcgggca ccatggcgaa cggcagcgcc gccgtggccg gtggattcac 1200

caccggatgc ccgtcatccc ccctccagct gagccccggc acgtcggcga ggctctcgcc 1260

ccggatgatg cgattcagca acgcgggcag cgcacgttcg ccctcaccgc tgatcacgaa 1320

gtcgagttgc tcgaaattcc ggtgcaacgc gggaccttgt gctccgtcgc agttgctgcc 1380

gccaaggacc gtgcggatgc ccggcgcgag tttcttcagc tccctggcca gtgcgagcga 1440

cgggacgttc tgcatgaagg tgctcgtgaa cccgaccacg tcgggaggat cggcagcgat 1500

ctcggccgcg agatcccgga tgaatccccg ggcgtacttg tgcatctcaa cgggaagtgt 1560

cgggtccatg tcccgctgct cgaggaactt cgcgtactcg tcgacctgat aactgtcgac 1620

gtcgtacagc gctggggtga acacccagtc ccctacgccg tggaagactt gatccgcgat 1680

gttcccgtag tcctcgcagg tgacggagcc gttgctctcc cgcatcaggt attcggccca 1740

gcggaggttg gcgtacagct catcgacggt ccagtcggcg gcgttcttgc ggacgcatgg 1800

ccccagtacg cccagcgcgc tggacggcgt gtcgagccct tgccacggca tggcgatcat 1860

caggagtttc ac 1872

<210> 3

<211> 1089

<212> DNA

<213> Actinoplanes sp.

<400> 3

tcagaaccag ttcccacggt ccgcccggcc gagcgccgcc aacagcgtcc gatgtgcctc 60

ggcctctccc actgagatcc gtacaccgtg ccccggaaag gctcggacct tgacccctgc 120

ggtagccgca gtccgggcga aagactcggc ggccgaagcg agcgggagcc agacgaagtt 180

ggctcgggaa agcaggacgg gcagcctcag ttccctgagt tccgcggtca gttcttcgcg 240

tgccgcagcc actgctgcca gacgttcaca cagttcgtcc tcgctgcgca gcgagagcat 300

tgcggcttgt tccgcgaagc gcgtcactcc gaaagggatt gccgtcttgc ggacggtggc 360

catgacctgc cgtggcccgg ccgcgtaacc gacccgtagg ccggcaaggc cataggcctt 420

ggagaacgtt cgaagtacca cggtgttgct gtgctcgctc agcaacaccg gcagacccgg 480

aggattggcg ccccggtcga actccacgta cgcctcgtcg aggaccgcga ccacatgagc 540

cggcagcgaa cgcaggaaac cgtgcagctc gtcttggtca atcacggttc cggtcggatt 600

gtgcggggag cacaggatca ccaccctggt ccgcgcattc acccgggtgc ggatctcatc 660

gagatcgtgg ccgccggacg cagtcagggg cacgtggact ccggtggcac ctgaaatggc 720

gaccaacagc ggataggcat cgaatcccgg ccagccatgg acgacttcgt cgcccttgcc 780

gcacagtgcg agaaggatct gctggagcac gcccgcgctt ccggggccga ccgcgacctc 840

atccggggag acgcacaagt gcccggcaat gtcctcggtc aggtcccgtg ctgtggggtc 900

ggggtaacga gcaagtcgcg gcaagccttt ttcgataccg gcaagcacgg taggcagcgg 960

ggggagaacc agctcgttgc tggacaggtc gaaggtgaac cgcgagctgc cttcggcgtt 1020

cgacgactcc ttgtcccggt aggcccgcat gtcccgcagg gtgctgcgtt ctgcgaatct 1080

cacgttcac 1089

<210> 4

<211> 957

<212> DNA

<213> Actinoplanes sp.

<400> 4

tcacgcctgc agactcctcg ccgtgcgtga ttcgcgggtg gcgagcagct cgtagagcat 60

ttcgtggacc ggcgcggtca ggcccgcccc gcgggcgagg cggaccacgg ccccggtcca 120

cgcttcgagc tctgacggcc gtcccgccag gatgtcccgt tgcagcgagg aggtgacgtc 180

gggcgactgc tggtccatga gctctgtcgc ggtgtccacg gcagctgccg gcagcgcgat 240

tccgagcttg atcccggtct cgtagatctc ccgcatgccg gcgatcagaa tgttgcgggt 300

gccggtgcgc gaccggagct cgccgatggt cgccccgccg gtggcggctc caaggctgcc 360

gatcgggacc accaacagga acttcgccca aaggccggcc cagatgtcgc tcggctcggg 420

cacggacacc gaggcagcac gcagcacctc gcgcagtcgt gccacccggt cggacacagt 480

gctgtcccac tcggtgaagg ccagagcgcc ggggggaccc acgtgcctca actcgcccgg 540

accggccgtc gaggccacga ccctgacgct gccggggagt acccgaccgc ggccgatcct 600

ggctgcgacc tgctcagggg cttccacccc gttctgcacc gtgaccacgg cagtgtgctc 660

gccgaccagc gggcccagcg cgtcgagggc cgccggcagc tgtgaggtct tgacgcagag 720

cagtacgaag tcgacctcgc cgatgtcctt cgggtcggcc gacgcccgaa cgtccggcac 780

acgtaagtca cttgagccgt tggtgatgcg cagcccctgt cgcctgagcg cggcgaggtt 840

ctcgccgcgg gccaggaacc gcacatcatg cccggcggcg gcgagcaggc cgccgaaata 900

gccgcctact ccgccggctc ccaccacagc aatgctcgga ccgccttgtt ccgtcat 957

<210> 5

<211> 1215

<212> DNA

<213> Actinoplanes sp.

<400> 5

tcatcgcggc aacctctctc ccactcgtat cgggagggcc tcgaggcccc ggctgagcga 60

actgtcggag agccaggcga cctcttccgg aggcacggcg agctcgaatc gcggcagtcg 120

gcgcaacagc gtggagaagg cgatctcgcc ctggagtcgg cccagcggag cgccgatgca 180

gaagtgggga ccgtggccga aggacagatg gcggttcggt gaccgcgtca cgtcgaaacc 240

gtccgggtcg tcgtacacgt cgggatcacg acccgcgctg gacagcgaca ggtgtacgaa 300

gctccccttg ggtatgtcca ctcccgcgag ctgcatgtcc tccgccgcga cccgcagcga 360

ggcccaggcg gccgaccctt cgtagcgcag gatctcctcg atcgccgaag ggatcagctc 420

gggggtggct cggagcatct cgagctgctg gggatggcgg agcaacagtg ctgtgccgtt 480

gccgatcatg ttggcgaccg tcttgtgacc ggcgatgatc agcaggagga gcgtcgaaag 540

gagctctgtt tcgctgtata cgcctgcgtc cctggccctg atgatctcgc tcagcaggtc 600

gtcccgcagg tccgtgcgcc gctcggccac gagcttggtg aaatagtccg tgaactcttc 660

gctcgcggct ttcagctccg cctcgtcgtg ctgcaacggg tcctggctga ggatgtagct 720

ccactccaga aacagcggcc ggtccgccac cggtatcccc aggtattcgc agatgacggt 780

gagcggcatc ggcagagcga aggaactgag caggtcgatt tcgccgttct ccggaaaggt 840

gtcgatgaga tcgtcgacga tgtcctggat gcgcgggcgt agttgggcga ccgtggccgg 900

caggaatgcc cggctgatgg gcttcttcag gcgggtgtgt ttgggcgcgt cggcgaatcc 960

caggttgccg accacgagga gactgctggc cacggccttg tctcgatagc gggcgggcag 1020

gttctcgact tgcttggaga ggcgcgagtc gccgagcgct tcctcaacga ctttgttccc 1080

gaggaccgcg tacgcgtcgg cgccgggggg aacattgatc cgatggaccg gacacttgga 1140

cctgtactcc gcggctgtcg cgtgcggatt cgaaccgggc tcggtgaaga attccgtggg 1200

aatcacgtcg gtcat 1215

<210> 6

<211> 303

<212> DNA

<213> Actinoplanes sp.

<400> 6

tcatcgtgac tcctcggctc gcgcggcttc gctgccacct gctaccgccg gcactatcca 60

gatgacgtcg tggtgctcca ctttcgtttc gaggccgtcg aggctcctga tgtcgctgtc 120

gttccggtag acattgacgt agcgcttcac ggacccttcc tggtccatga gtcgctcgag 180

gacccccgga caggtctggt cgagaccgac aaggacctcc cggatattgg cgccctcgac 240

aggcaactgc cgccgaccgc cggtcaggac gtggaaggct gcgggaagtt tgacatcggg 300

cat 303

<210> 7

<211> 717

<212> DNA

<213> Actinoplanes sp.

<400> 7

ttattcctcc tcgagtagga cttcgaactg ctcatggatc gccgccggaa tccgtatgtc 60

aagcctgtcg cattgctcgg caccgggccg ccaggccgct acccgatgtg cgataccgcc 120

actactctca tgcacgtacc agatgacgtt cactggccag catgactgcc ggaaaaggta 180

ttcatccatc tgtgtcggat gttcgctcag gcgctggcgc cgttcgtgcg gcgggccgat 240

ttcatgccag ttcggatgtg aatgtatgga gcccagcaac tccaggccgt tcgccgactg 300

ttgcctgatc gcctgcaaga cgccctgttc atcggaccag aatcctcgcc ccggattctt 360

gtacacgtcg ccgaactgcg gggcgatcgt cgcttcgaat tcggccatga cactctcgtc 420

actgtcccgg acattcggca cgaattccac atcgctgatc actatttcag cgccgcccac 480

ctgcccgaac aacagacccg atgcacgcgg cagatggctc gggacgccat cggcgtcgcg 540

gctgtccagg catttttggt actcccccag tgcgctgctc aggaaacgcc tgacgggttc 600

gatttcaaat cggacggtca ccggcagatc gtcgagcagg gctgcactgt ctgctgtgcc 660

acccagaccg tcatccgtag cagctgtcac atactgggca tctttcacgc ctgccat 717

<210> 8

<211> 1491

<212> DNA

<213> Actinoplanes sp.

<400> 8

tcatgggcgt acgccccctg cgctctggcg aggcaggtca gtcacccctg gcatacgagg 60

gccgcggagc gtggaggtct tgatgccgac ccagcacaac agccctccgg ccgccgctac 120

ggcggcagcg atccagaagg cggtggtgaa gccgtcgcgc gccggcgtat cggaaccgga 180

gatatgccag gcagccagca aggcgacagc cagctgactg cctacgacgc cgcctgccgt 240

gcgcaccacc gtgttgacgc cattggcggt tgcggtgcgt cgggcctccg tcaagtcgct 300

gatcaccgac gggagtccgc tcagcaccag tccggaaccg agacctacga ccaggtaacc 360

gaccgccact tgccacccgt tggcattcca cgcccagagc gagatcgccc cgaccgccat 420

cacggcgaag cctgatgcga gcgtcgcgcg tacggacgtc aggcgctgca gcagacctgc 480

tagtggtccc gcaggcaaca gcaccaatga gccgggcaga aggagcagac ccgccatggt 540

gacatcggcc ccgagcccgt agccgatggt ccctccgccc ggtagccgct gatccgccgc 600

ggtctgggcg tacgtcggca gcaggacgta gaacacgaac gagaccacgc cgaacacgaa 660

cgccgcaccg tgcaccgaga cgaaggagcg accggccacg accgcggggt cgatcaaggg 720

ggcgggcgac tttcgttcga ctacgaccag caatcccagg agaaccgcgg aggccccgaa 780

cagagccagc gttcctgtgg acgcccaccc ccatgaggtg cccttggtca acgcgagcaa 840

cagcgcgacg aggaccaagg ccaggagcac cgccccgggc acgtccaccg gttcgccggc 900

ttcgccccgc tggtccggaa cgtacttcgc aacgagcccg atcgcgccca ggatcagtac 960

cgcggcgacg gcgaacagcc agcgccatga ctggtggtcg acgaccagac ctcccacgac 1020

caggccgatg ccggccccca caccgatggt ccctgacacc agcccgaggc ccgaacgcag 1080

ccgctgctca ggaagtacgt cgcgcaggat gccgaaggac agggggatgg ccgcgaggct 1140

gactccctgc acggcacggc atgcgatcag aaccccgata ttgcctgcca cagcacaacc 1200

gaccgtgccg atcaggtagg tggtgagaac cagcagcaaa actttgcgct tgctgtagcg 1260

gtcccccagc cggctcagca agggcgtgct cgccgcactg gtcagcagga acacactcag 1320

gatccacgcc gaccacgtcg aggccgtgtg taactgcacc tgtagtacgt gcaaggccgg 1380

gacgaccatg gtctgcatga gtgcgtacga cagcacggag acgccggtgg cgatcagcgt 1440

catcgtcgcg ctgatcgacg gtggacgtgg cgaagtgccg ttgtaggaca a 1491

<210> 9

<211> 942

<212> DNA

<213> Actinoplanes sp.

<400> 9

atggaactgg atttgcggca cctcaggtac ttcgttgccg tagccgagga aggcgggttc 60

acgcgagccg cggcccgcct gcacatgaca cagccgccgt tgagcgtggc gattcgtcaa 120

ctcgaaagag agctgggtct ccagcttttg gacagaacgg gcaacagagt cgaactcacg 180

tcggtcgggc gcgacttcct gactcacgcg aggaacttgt tgcagcagtg gcaggtcacg 240

gtcgagagga tgcggcaggc ggggtcgcag gatgtcgaac ggctcgtcgt cgcgttccgc 300

ccggccgtca gccgccctct ggcacaccgg accattgaac tcatccgcga aaagcaccct 360

gagtatcagg tagtgccccg gtacgtaccg tggaccgaac agacagcatg cctggaggca 420

ggggacgctg acgtgtcctt cgtgctggag cccgcggact acgtgggcct cgagagggcc 480

accgtggccc tgttaccccg ggtcgtctgt ctgccatcgg ctcacgagct ggccagtcgt 540

gactccgtgt cgatcgacga cctgagcgag gttccgatca ttcgccccac cggcgggtcg 600

cccgagtggt ccgacttctg gggtggtgag gtgtgccccg gcaagcgcac ctggaaggaa 660

cctcccacag cgacgcgcct cgacgaggcc atcgacctcg tggccctcga gaacgcagcc 720

gcgctcgtcc ccgtctctgt catggcagtc cagcaccgtc aggacgtcgt cttcatccct 780

gtgacggatg tgcctgccgc ccggttgtcc cttgcctggc gtgagggttc cgactccgaa 840

ctggtacgcc tcgccgtcag gtgcgctcag gccgcagccc aggatccagc cgtcaggacg 900

ctcttcggag aacctcgacc aaccggaacc gctccggcct ga 942

<210> 10

<211> 903

<212> DNA

<213> Actinoplanes sp.

<400> 10

gtgcgtcgtt gggcagcaca gtcgggcccc gaagacctgt atttcgtcag caacctgcac 60

gccatgacga ccaagcacga ccccgaacgt ctccaggaac tgaccgacca ccaactcgct 120

ttactcatcg cggcgggcgt accccaggaa cgtctcttcg tgcagtcgga cctcatccag 180

gagcacatgg cgttgacgtg gcttctcgag tgcacctgca ccttcgggga ggctcgaagg 240

atggtgcagt tcaaggagaa gtcccagggg agcaactccg tacgccttgg tctgctcacc 300

taccccgtcc tcatggcggc ggacatcctg cttcatggcg cttcagaggt gcccgtcggt 360

cacgatcaga accagcatgt ggagctggcc cggaccttgg cgcggcggtt caacacggac 420

tacggcgagg tgttcacggt tccgcaagcc gtcctgcccg tagccgcagc ccgggtacgt 480

gatctcgctg cccctacgcg gaagatgtcg aagtcgtcct cggacggcag cggcatcgtc 540

tacgtcctgg acagcccgga ggccgtacgc cggaagttcc aacgcgcagt gacagacgga 600

gaaaacaccg tccgctacgc cccggacgaa cagccgggcg ttgccaacct cctggagatc 660

agggctgcct gcactgacac gctcccgagc gatgcggcga agggtatcga ttcctaccgt 720

gacctcaagg aagcagccgc agaggcagtg atctccctga tcgcaccggt gcgtgagcgg 780

gcactgcagc tcctcgaaga gcgatcggag ctggcgaaga tccgggctga gggggccgac 840

cgtgctcggg cgcggtcacg agaccgcttg gatcgtgcgc tcagccttgc cggtctgaag 900

tag 903

<210> 11

<211> 795

<212> DNA

<213> Actinoplanes sp.

<400> 11

atggctgacg atcccctggt catcggtggt acgagctact cgtcgcggct catcatgggc 60

accggcggcg cccccagcct ggacgtgttg gaacggtccc tggtggcgtc cggcaccgaa 120

ctgaccaccg tcgcgatgcg ccgcgtcgac ccgagcgtga agggctcggt gctctccgtc 180

ctcgaccggc tcggcatcca ggtgctgccc aacaccgcgg gctgtttcac cgcgggcgag 240

gccgtcctga cggcccgcct ggcccgcgag gcgctcggca ccgacctggt caagctggag 300

gtcatcgccg acgagcggac cctcctgccc gatccgatcg agaccctgga ggcggccgag 360

acgctggtcg acgacggctt cacggtgctg ccgtacacca atgacgaccc ggtgctcgcc 420

cgcaagctgc aggacgtggg ctgcgcggcg atcatgccgc tcggctcccc catcggctcg 480

ggcctcggca tccgcaaccc gcacaacttc cagctgatcg tggagcacgc gtgcgtgccg 540

gtgattctgg acgcgggtgc gggtacggcg tccgacgcgg cgctcgccat ggagctgggc 600

tgcgccgcgg tgatgctggc ctcggcggtc acgcgcgcgc aggagccggt cctgatggcc 660

gaggggatgc ggcacgcggt ggaggcgggg cggctcgctc atcgcgcggg ccggattccg 720

cgccgccact tcgcggaggc gtcctcgccg accgagggca tggcccggct cgacccggaa 780

cgtccagcct tctga 795

<210> 12

<211> 1179

<212> DNA

<213> Actinoplanes sp.

<400> 12

gtgtcgctgc cacccctggt cgagccagct gctgagctca ccgtcgacga ggtccgcagg 60

tactcccgcc acctgatcat cccggacgtc gggatggacg ggcagaagcg gctgaagaac 120

gccaaggtgc tctgtgtggg cgcgggcggc ctgggctcgc ccgcgctgat gtacctggcc 180

gccgccggcg tcggcacgct cggcatcgtg gagttcgacg aggtcgacga gtcgaacctg 240

cagcgccaga tcatccacag ccaggccgac atcggccgct ccaaggccga gtcggcgaag 300

gactcggtcc tcggcatcaa cccgtacgtg aacgtgatcc tgcacgaaga gcggctcgag 360

gccgagaacg tgatggacat cttcagccag tacgacctga tcgtcgacgg cacggacaac 420

ttcgccacgc gttacctcgt caacgacgcc tgcgtgctgc tcaacaagcc gtacgtctgg 480

ggctcgatct accgcttcga cggccaggcg tccgtcttct ggagcgagca cggcccctgc 540

taccgctgcc tctacccgga gcccccgccg cccggcatgg ttccgtcctg cgccgagggc 600

ggcgtgctcg gcgtgctctg cgcgtcgatc ggctccatcc aggtcaacga ggccatcaag 660

ctcctcgcgg gcatcggcga cccgctggtc ggccgcctga tgatctacga cgccctggag 720

atgcagtacc gccaggtcaa ggtccgcaag gacccgaact gcgcggtgtg cggcgagaac 780

cccacggtca ccgagctcat cgactacgag gcgttctgcg gcgtcgtctc cgaggaggcc 840

caggaggccg cgctcggctc cacgatcact ccgaagcagc tcaaggagtg gatcgacgac 900

ggcgagaaca tcgacatcat cgacgtccgc gagcagaacg agtacgagat cgtctcgatc 960

cccggcgccc ggctgatccc gaagaacgag ttcctgatgg gcggcgccct gcaggacctg 1020

ccgcaggaca agaagatcgt cttgcattgc aagacgggtg tccgcagtgc ggaagtcctc 1080

gcggtcctga agtctgcggg cttcgccgat gctgtgcacg tgggtggcgg cgtgatcggt 1140

tgggtcaacc agatcgagcc gagcaagccg gtgtactag 1179

<210> 13

<211> 1275

<212> DNA

<213> Actinoplanes sp.

<400> 13

tcagcttgtc gtggcggtgg cgccggcggt tccgccgctg accagcttgg cccaggtggc 60

ggggccggcg atgccgtcgg aggtcaggcc cttggccttc tggaaggcgg tgagcttgct 120

ggtggtggcc gggccgaaga cgccgtcggc ggtgacgtcg tagccgttgt cggcgagctg 180

gcgctggagg gcggtgacgt cggtgccctt cgagcccgac ttcaccgtgg cgatcagctt 240

cgcccaggtg gcgggaccga ccatgccgtc ggcggcaagg ccctcggcct tctggaacgc 300

ctggaccttc gcggctgtcc ccgtaccgaa cacgccgtca gcagtcgtcg cgtacccgtg 360

cgcaccgagc agcaactgca cggtcgccac gtcgacgccc ttgtcgcccg ccttcaccgt 420

cggccacgac gtgcccggct gcggctgcgg cgcaccgcct cccttggcga gggcccgcag 480

ctggtccagg ctgccccggt acacgttgcg gtcgcccttg cccgcggcgc cgttcttgcc 540

ggggaggcct gggatcgcct cggactccgt gtactgccag agcgaccagg cacccgcgcc 600

cggcacgtcc tgcggctcct tcgacccgct ttcgtagcgg gccagccaca gcgggtggtc 660

cttgaagacc tggcccttgc cggccatgca gccgttcacg aacgacgccc gtgtgtagac 720

gatcggcgtc accttgaacg cctcctccac gcggttcagg aaggcggtga gctggtcggc 780

gcggagcgcc ttcgggcaca cctccttgcc gttcacccac gtgccctcga cgtccagaac 840

cggcggcagc tccccggccc tcttgccggt gtagccggcg gaccgggccg cgcggatgaa 900

gtggtcggcc tgcgcgccgc cgtccgtggt gctcttcggg tcgaagaagt ggtacggggc 960

gcgcagcagc gatgtgccgg acgcgtcctt gaagtcccgt gcgaaccaag ggtccttgta 1020

accggtgccc tgcgtcgcct tgaggaacgc gaaggagttg gactgggcga cgcgcttcca 1080

gtcgatgggc ttgccggtcg cgtcgtggtt gtggtggctg gtgtcgacgc ccttgacctc 1140

gtacgtactg ggaggggcgg ccacgctctc gtccgcggtg gacagcagga caccactcat 1200

gagcgacgcg gtggcgacgg ccgccgcggc caggcgcagt ccgtgacgac ggccgcgcgg 1260

gttctccatg gacac 1275

<210> 14

<211> 996

<212> DNA

<213> Actinoplanes sp.

<400> 14

atgggcgttg aacgcgacga gccgacgcgg tcagcgaggc gggaactcgc cctgctgttg 60

cggagctggt gggaggcgca ccccgacaag atcacgcagg aggcgctggc ccggcggatc 120

acggagcggg gcgtacggat cagccaggag atgctgtcgc gctacctgaa ccggtcccgc 180

ccgaccacgg cccggcccga cgtgatccgc accatgcacg aggtgctgcg ccgggcgccg 240

gaagagctgg acgtggccct ggaactgcac gctcgggcca ccgccccgca gacgccgccc 300

gccgaggggg cggccacgag ccagccggcc ggggacgcgg ggaccgccgc gccgaagggc 360

gtagagccga cctcggcggc cccgctcttg acccgcacgc cccacacgcc ccgccccgct 420

tcgcggaaga agtggccgtg gatcgccgtc gtcgcggccg cggtcgtcgg cgcgtccggg 480

ctcaccgcct tcatgacact gggcgaccag cggcagaaca ccccgcgggg acacggagcg 540

acaccctccg cctcacccac cgccctggtg tcacccaccg cccaggggtc gcccgccggc 600

acgcatcctc ccgcggagtg ccgcgacgag tcctgcttcg gcatcgacgc caagtacgcc 660

atctgccagg acgacgccgc cacttactac acgggccgcg cccacggcgt cctcgtcgag 720

ctgcggttca gccccgcctg ccaggcggct tgggccaaga tgagcggcac ctcgcagggc 780

gatgtcgtac gcgtcaccaa caacgcgggc cgcagccgcc actacaccca gcagtggggc 840

cgcgacgccc acaccacgat ggtggaggcc gtgagccccg acgacgccaa ggcttgcgcc 900

cgcaccccgc gcggcgaggt gtgcgccacg aaggccgtcg cgtccgcccc gcgcgacgcg 960

gcacctggcg agcgcgcggc acctggcggg cgctga 996

<210> 15

<211> 1041

<212> DNA

<213> Actinoplanes sp.

<400> 15

atgcccgcac gcaccacacg aaccgcacac accacacgca ccggccggtt ggccgtcgtc 60

gccctcgcgg ccttgacctg tgcgggcctg gtcaccggaa ctgcagccac ggccaccaca 120

cccgactccc tgcccaccgc gaagcgcgcc gcagcgcccg acgcagcggc tgtatcgtgg 180

ccgacgctga aggcgggcgc gcgcggtacg gaggtgaccg cgctccagca cctgctgatc 240

gcccgcggcc aatccgtcgc cgtggacggg gagttcggcc cggccaccac cacggccgtc 300

aaggcgttcc agaaggccga cgggctcacc gccgacggca tcgtcggacc cgccacctgg 360

gccaagctcg tcccgacgct gcgtcagggc gcgcagggcg cggcggtgaa ggcggcccag 420

accctgctga agacccgtgg ccaatccgtc gccgtggacg gggagttcgg ttcggccacc 480

acctcagccg tcaaggcgtt ccagaaggcc aaggggctca gcgccgacgg tgttgtcggc 540

acgcagagct ggtccgcgct cctcacctcg gactccggcg cgccgtccgg gaaccgggcc 600

gcgttcgccc agcagatcct caacaccagc ggcatcgagc tggcgaccgt ccaccccggc 660

ggcacccacg ccggctccac cgcccggcag aacatcatcg acacagccaa cggcaagggc 720

gctctgacca gtccctggag cgacaagccg aaccagcgcg tggcgctcga cacccggatg 780

ctcaacgggc tgctgaagct gctctcccag gacggctacc ggatctctgt ctccgagatc 840

gtcggcggcg accacagcac gaactcccgg cactacgcgg gactcggctt cgacatcaac 900

tacatcaacg gccggcacgt cggcgagagc gccccgcacc agggcttgat ggccgcgtgc 960

cggaagctcg gggccaccga ggtgctcggt ccgggcgacg ccggccacag ccgccacgtc 1020

cactgcggct ggccgcgctg a 1041

<210> 16

<211> 723

<212> DNA

<213> Actinoplanes sp.

<400> 16

ctactccgta gcaccgtccg atttcttgcc ggctgcggcc aaggttcctc ccaatagggc 60

cgcgcccagg ggccatgaac gcatgtgcac ggggagggtc acgcagtgat ccccgacgcg 120

catcacaagg cccttgttca ccaaatcgcg tgcggcggcg gcgaattgcg agtttcgggt 180

cacctcggcc cacgcgcatc cctccaggcc caggaggaag acggccatct gggtgggatc 240

gtccacgatg atttcgcggc tggattcggg gcgctgatcg acgacggaca ggaacttcgg 300

gcccttacgg aagtagagga ggccgaaatt gctggaggac cgccactcac cgacggacgg 360

atgccctgtc tccagcaccg tgatgctgtc gggggcagga aggtgatcga gacgggggac 420

caggtcgagc tgttcggcgc ccagggtcag tgaccaggtg actctggtgc cgatcgagga 480

acattcgcga atcaacgcga tgatccgcac gatcacgtga ctggggagtg ccgagaagtc 540

ggcgggctcc ggcagccgta ccccggcgac gggcacccgg tgcagtagcg ccgccagttc 600

ggtggcgacg ggaagttcgg cccggtcgag tccgagggtc agcgtctcgg tcgcgggacg 660

gccaccggcc ggtacccgtc cctcgtccga atctcgggcc ttgagttcat cgatgtccag 720

caa 723

<210> 17

<211> 2541

<212> DNA

<213> Actinoplanes sp.

<400> 17

gtgtcggcgg tgctgggcgt tcttctcgcg atatcactcg caactgcccc cgcccatgcg 60

gcagttcgct cggccgcggc ggtcgatgtc tgtcggtcgg ccgccctgag caaggcgcgt 120

gtgagcacgt gggtgcggct tgagcaccgc gatggtacgt acagcaggat ccgcagcgag 180

ctcagcgtcg aggtgcccga ggattggccg ttggccaagg acctgctgct gagtgaggac 240

agccgccggt acgtcgcggc gatgtcctgc ctcacccgta ccgatcgggg ccggcaacgc 300

cgctggtcgg agtggaggag cagccgtccg acggtggcgt ccacgaagag cggtggggtg 360

aaggtcgtcg accgtacgca ctcctgggtc aacgtgtatc gggcgcacat cgatgtgggt 420

acctggcggg tccgtgcggg tgcggagcgc tggaccgtac aactgcaagc tccgtccgcg 480

ctgaacgcgg cccgctggga tgagatcagg gtggaacccg gcgccccggg agccgagtcg 540

gcgaccccgc ggcctgacga ggggcgcggc gccacggcgt tggtgtggca tccccagaac 600

caccgtgaga aggcggctgc tcctgccgtg agcgttgcgc tcaagccctc ctggcagcgt 660

tcgtgggcag cccagaacga ccggctggtc gccgtggcgc tggatcgggg cggatggctg 720

ctctgggacg cgacgagtgc cgccctgttg ctgtacgcaa ccgtcctgta ccggaggcgt 780

tccgctcctc ccactcaggc tcaggagcgc acactgcgca atctttccct gtgggccaag 840

gccctcgtgg tgctggtcgc gctgacgagc atggacgacg tgctcattcg gtacgtgcaa 900

cggcggggcg acgggctgtt gctggacgag cagatcccgc gcgggaatgc gttcgccctg 960

gcagccgtca tcgtgctgtt ctgcgtcggc aggccgcgtc ggcggatctg ggcggcggct 1020

gctgtgctgg ccgtgccgac ggtggctgcc ttgccgcagt ggttcgaact ctccccgcag 1080

cgcttcgtgt ccgacgacga gtgggcagtc acgttggcgg cccagggggt cgccgcctgc 1140

tgcatgctgg ctctcttggg gctcggcttc gtaactgccg cctggcgctt ggccgttgac 1200

ggggacctgc tgccgatgag ccgtcggcac ccggggcacg cccgggtcct caggctccgc 1260

atcgccgggc cggtgatcct ggtgtgtacg gccgctgtgg cgatctgttt cgccctggcc 1320

caggagcgca actggcagcg tgccacctgg ctcagcgatc gctcggaccc cgcctacgcg 1380

accggccagt ggagcgatcg cgtgtgggag gcggtgtggt ccgtcgccaa tgggcaggac 1440

tggctctcgt ggcaggcctg gctgctcacg ggagttgcgg tgcttgcggt cttgcgcacc 1500

tggcgcgccc cggcctccgt ctcccctctg gacgacccgg cggaccgcct tctgttcctc 1560

gccttcttcg ccatcgtggc cgcggcttcc ggcggctact ttctgggcaa cgaggtgctc 1620

accggcttgt ggattccgct cagcatgctg gctctctact gggtggtggt tcccttcacc 1680

caccgctcgg tactggcgca gcctttcgag cggtccgggc ggcccctcgc cgattccgcg 1740

gggcccggcg cacgcaccgt actgcttgcc aaggcccgct cctaccgcga gacccatgcc 1800

gaactgcgcc gcctcgacca ggggttgttc ggggacgtgc caccgaagcg aagcgacctg 1860

gaacaggagt tgagcgacct gcacaactgg cccacggcag gtggctccga ccggcttccc 1920

gccaaggtgt ccgtggtgga cggagcactg gcgctggggc cacgagacac ctggtgggcc 1980

aatggcagcc gctgtgcccg cctcgccttg gttccggcgg taccggcggc cctgctcctg 2040

gcctgggtct ggaaggtcaa gggcgaggcc tggcacgcga ctctgcacga acagttcggt 2100

ctgccggatg tcctgctctt gttcgtcggg gagatggtga tgttcaccag ctcggcgttc 2160

gtcctgggcg cgctgtggcg ccatctgcca gggcagcgcg gcgccgccaa ggccctgccg 2220

gtgacactcg ccttcgcgct gcctatcggc ttggacgcgc tcgtctaccg gttcaccggc 2280

gagagcaccg cgaacctcgc tctggctgtg tcggcgatgc tgttcgtgct gactgtcacc 2340

agcatcgctc tcgacttcga cacgttccgc ggcgaacggc gttactggca gagccggttg 2400

ggcctgctcc tttcgatcta tcagatgcgt tactactcgc tgcaggccgc ctacctgatc 2460

gcccaggtcg ttgccatgat cacgatctgg gagttcttcg cggaacccga cgtggtgccg 2520

aagccctccg actcgaagtg a 2541

<210> 18

<211> 1647

<212> DNA

<213> Actinoplanes sp.

<400> 18

ttacgggcgc cactggtcct ggctttgcgg tagtccttgg taggtgccgt agtccgttga 60

gccttggtgg gtgccgtagt ccgttgagcc ttgctccatc gtggggtgct ccgtcgtgga 120

gtgctccggc gtggggagct ccgtcgtagc gtgctccatc gagccgatcc aggcgcgcgc 180

cggggggcgt ccgacgtact tgccgaagag cagtgccgcg gcggtggcag cgagtacgcc 240

ggggaccatc tcgtagacgc ccgattccag cggcccgaga agcgggtcga tgtacttcca 300

caggaacacg gtgagcgcac ccgtcaccat gccggccatc gccccggctg ccgtcatgcg 360

cggccagaac agcgacagga tgatcaccgg gccgaaggcc gcaccgaatc cggcccaggc 420

gtacgcgacg atgtcgagca cggcgccgcc gctcagcgcg atcgcatagg cgaccaatgc 480

cacggccacc acgctcagtc gtccgaccat cagcagcaac gtgtcggagg cccgccggtt 540

gaggaacgcc cggtagaagt cctcggtgag ggacgtggcc gagaccagca gctggctgtc 600

caccgtggac ttgatcgcgg ccagcacggc caccagcagg attcccgcga tccaggggtt 660

gaccaggtgt gtggacagct cgatgtagac ggtctccggg ttgtccagcg gctcgtcgag 720

cacggcgatc cccgcaagcc cgatgagcga ggaacccccc agtacgacga ccacccagcc 780

cacacccaga cggcgggcca gcggtatgtc ctttgtgctg cggataccca tgaagcggat 840

caggatgtgg ggttggccga agtagccgag cccccaggcc aacagcgaga tcatcgcgat 900

ggcgccgagc ggctcgccgg ccgaccacgt gttgccggcg aaggatgcct cggccaccgg 960

gtcgagtagt gccggggtct tgtcgctgag cgcgtcgtgc agcgcgccga agccgccgag 1020

ccgccagaga ccgagcgcgg ggaggacgag tgccgcgagg aacatcagcg tgccctggat 1080

ggagtgcgtg atgctcacgg cccggaagcc gccgaggatg gtgtaggcaa cgatcaccac 1140

ggcaaatacg gtgagcccga actcgaagtc ggcgccgaat atctcgttga acaggagacc 1200

gccggcgacc agcccgctgg cgacgtagac ggtgaagaac aggaccgtga cgatggccga 1260

gagcagccgg agcatcctgc tccgatcctc gaaacgttct tccaggtacg acggcagggt 1320

cacggagttg ccggccagct cggtgtaggt gcgcaagcga ggtgcgacaa accgccagtt 1380

gagataggtg ccgacgatca ggccgacggc gatccaggtg gcgccgatcc cggccatgta 1440

cacggcgccg ggcagaccca gaaacaacca gccggacatg tcgctggcgc cggcagacag 1500

ggcggccatc ggggcggtga gtcggcggcc gccgaccgtg aagtccgcga atgtggccgt 1560

ttccttttgc gtcatgacac cgatcatgac catcgcaatc agaaagaccc cgaaagtgat 1620

catggctggg acggtcaggg tgagcat 1647

<210> 19

<211> 240

<212> DNA

<213> Actinoplanes sp.

<400> 19

ctacccgacc gcctcccccc atgggccttg agcgatgccc ggtgaagcgg ctgccccggc 60

atgcccttcg gtggtggcgc tcctgttgac aggattcgta tgggcagtgc acttcggctg 120

ggcagtgcag ttcgacgatc tcgtagacgt atggccgttg cctgtggcgt ggtcatcgcc 180

gtggctggtg ggctcctgtg ccccgttgtc gccgcgccga ccgccggtgc ggcggaccat 240

<210> 20

<211> 1956

<212> DNA

<213> Actinoplanes sp.

<400> 20

gtgaccgtgg acacccccac ccgtgccgcc aaggagaaac ttgccgggct cgggctcgac 60

ctgaccgagc atgccgggca tggctttgtc gaagtcgtgc tgcacagccc ggccgacgcg 120

ctcgcgctgc aagtgggcgg attcagctgg aaggttcgcg tacccgatct cgtccagcgt 180

gagtccgacg tgaacgccgc gaaccgggcc tatgccgccg ccaccggcac ctcgccgctg 240

ccgtccgggc gggacagcta ccgccggctc gccgactaca acgacgatct cggccggatg 300

gccgaccaga atcccggact cgtacggaag ttcacgctca agcacaagag cctcgaaggc 360

aagcccgtgc acggggtgga gatcacgcac gacgtcacgg ctgtcgacga cgggcggccc 420

gtcttcctga tgatgggcct gcaccacgcc cgcgaatggc cctccggcga gcacgccatc 480

gagttcgctc atgatctcgt caggaactac gggagcgatg agcggatcac ctcgctgctc 540

cagaaggcgc gggtgctcgt cgtgcccgtc gtcaacgtcg acggctttga aaagtccgtc 600

aacgatgggc agttgatcga tctgcgggag atcgacgacg gcggcaccgg atcgatcctc 660

gccacgcccg gcaacgccta caagcgcaag aactgccgga tcgtcgacgg cctgagcccg 720

gtcgcgggcg agtgcgcgct ggcgagcagc cccggcgggt tcggtgccgg tgtcgatctc 780

aaccgcaact acggcggatt ctggggcggt cccggcgcgg ccgccgagtc cgtgcaggcc 840

acgtaccgcg gcgccgcgcc gttctccgaa ccggagacgc agaacatccg cgagctggtc 900

agcagccgcc aggtgaccgg cctgatcacc aaccacacct tctccaacct ggtgttgcgg 960

ccgaacgggg tcgcgcccga cacggtcggt ccagacgggc agcccatcgg caacccgccg 1020

gacgaggccg cactgaagga gctcggcgac cggatggccg agcagaacgg ctatacgagt 1080

caacacagtt gggagctgta cgacaccacg ggcaccaccg aggactggtc gtacaacgcg 1140

acgggcggct acggatacac cttcgagatc gggccccacg agttccatcc gccgttcccg 1200

gaggtcgtcg acgagtacgt gggcgcgggc gagtacgccg ggaagggcaa ccgtgaggct 1260

ttcctgctcg ccctcgagag tgccgtcgat cccgagtcgc actccgtgat cagtggcaag 1320

gctcctgccg gggccacgct gcggctgaag aagacgttcg ccacgcccac ctggtcgggc 1380

acgatcaagg acaccctcga caccacgatg accgtcggca gcggcggcag ctacacctgg 1440

cacgtgaacc cgtcgacccg gccggtcgtc aaggcccgcc agatcgaggt catcggctcc 1500

gagccgctga agcggcagac ctacacgggc acgaccgcgc ccggacagcc gacggagcag 1560

gagttcgtcg tcgaccggga cgccgacgtc ttcgaagcga agctcgactg ggccacgccc 1620

gacgacctcg acctgtacgt cctgcgcaag aacgccgacg gcagcctcac ccaggtcggc 1680

agttccgccg gttccgtcgg cgagaaggag cgggtcctcc tcgacgaccc ggagcagggt 1740

acgtacgtac tccgcgtgga gaactgggct tccgtcgccc ccagttggac cctcaccgcg 1800

tccctctacg acgccaccgt ggacgagatc ggcggcgtca tcgagaactg gacgctctcc 1860

tgcgagaagg acggaaaggt gcttcagcag gtgcccgtcg tcgtcgaccg tgggcagcgg 1920

gtcaaggcgg acttgaagaa ctgcgcgaag ggctga 1956

<210> 21

<211> 623

<212> PRT

<213> Actinoplanes sp.

<400> 21

Met Lys Leu Leu Met Ile Ala Met Pro Trp Gln Gly Leu Asp Thr Pro

1 5 10 15

Ser Ser Ala Leu Gly Val Leu Gly Pro Cys Val Arg Lys Asn Ala Ala

20 25 30

Asp Trp Thr Val Asp Glu Leu Tyr Ala Asn Leu Arg Trp Ala Glu Tyr

35 40 45

Leu Met Arg Glu Ser Asn Gly Ser Val Thr Cys Glu Asp Tyr Gly Asn

50 55 60

Ile Ala Asp Gln Val Phe His Gly Val Gly Asp Trp Val Phe Thr Pro

65 70 75 80

Ala Leu Tyr Asp Val Asp Ser Tyr Gln Val Asp Glu Tyr Ala Lys Phe

85 90 95

Leu Glu Gln Arg Asp Met Asp Pro Thr Leu Pro Val Glu Met His Lys

100 105 110

Tyr Ala Arg Gly Phe Ile Arg Asp Leu Ala Ala Glu Ile Ala Ala Asp

115 120 125

Pro Pro Asp Val Val Gly Phe Thr Ser Thr Phe Met Gln Asn Val Pro

130 135 140

Ser Leu Ala Leu Ala Arg Glu Leu Lys Lys Leu Ala Pro Gly Ile Arg

145 150 155 160

Thr Val Leu Gly Gly Ser Asn Cys Asp Gly Ala Gln Gly Pro Ala Leu

165 170 175

His Arg Asn Phe Glu Gln Leu Asp Phe Val Ile Ser Gly Glu Gly Glu

180 185 190

Arg Ala Leu Pro Ala Leu Leu Asn Arg Ile Ile Arg Gly Glu Ser Leu

195 200 205

Ala Asp Val Pro Gly Leu Ser Trp Arg Gly Asp Asp Gly His Pro Val

210 215 220

Val Asn Pro Pro Ala Thr Ala Ala Leu Pro Phe Ala Met Val Pro Ala

225 230 235 240

Pro Gly Tyr Asp Ser Tyr Phe Gln Ala Leu Glu Arg Ser Pro Val Arg

245 250 255

His His Val Arg Pro Met Leu Val Leu Glu Thr Ser Arg Gly Cys Trp

260 265 270

Trp Gly Glu Ala His Gln Cys Thr Phe Cys Gly Leu Asn Gly Ser Asn

275 280 285

Ile Asp Phe Arg Ser Lys Ala Pro Glu Arg Ile Ala Gln Glu Val Arg

290 295 300

Glu Leu Ala Glu Arg His Gln Ile Leu Asp Leu Val Met Val Asp Asn

305 310 315 320

Ile Leu Asp Met Lys Tyr Leu Asn Thr Ala Met Pro Glu Ile Ala Ala

325 330 335

Leu Asp Cys Asp Leu Arg Ile His Tyr Glu Ile Lys Ser Asn Met Asn

340 345 350

Arg Glu Gln Leu Ser Arg Leu Lys Glu Ala Asn Val Leu Phe Val Gln

355 360 365

Pro Gly Ile Glu Ser Leu Ser Ser His Val Leu Arg Leu Met Asp Lys

370 375 380

Gly Val Ser Ala Ala His Asn Val Arg Met Leu Arg Asp Gly Gln Asp

385 390 395 400

Leu Gly Leu Asn Val Thr Trp Ser Ile Leu Tyr Gly Phe Pro Gly Glu

405 410 415

Thr Glu Asp Asp Tyr Arg Gly Leu Leu Lys Lys Leu Ala Thr Leu Glu

420 425 430

His Leu Glu Pro Pro Thr Gly Ala Trp Arg Ile Ala Leu Glu Arg Phe

435 440 445

Ser Pro Tyr Phe Glu Asp Pro Thr Gln Gly Phe Met Phe Arg Arg Pro

450 455 460

Ser Glu Ile Tyr Asp Phe Ile Tyr Gln Ile Pro Gln Asp Gln Leu Tyr

465 470 475 480

Asp Met Val Phe Phe Phe Asp Thr Ser Val Arg Gly Ile Ser Gly Pro

485 490 495

Ile Glu Asp Glu Met Lys Gln Ala Cys Glu Glu Trp Ala Lys Ala Tyr

500 505 510

Pro Gln Gly Thr Leu Ser Tyr Trp Thr Asp Asp Arg Gly Arg Val Val

515 520 525

Ile Glu Asp Arg Arg Ala Ser Trp Pro Thr Glu Val Ile Glu Leu Asp

530 535 540

Glu Val Arg Ser Asn Val Tyr Leu Gly Met Phe Gln Cys Ala Ala Arg

545 550 555 560

Glu Gly Ile Arg Arg Arg Leu Ala Asp Ser Gly His Val Val Gly Glu

565 570 575

Ala Glu Leu Glu Glu Met Leu Arg Tyr Phe Val Asp Arg Gly Leu Ala

580 585 590

Phe Glu Asp Glu Gly Arg Tyr Val Ser Val Ala Leu Gly Val Asp Pro

595 600 605

Tyr Arg Arg Lys Leu Val Gly Gly Lys Glu Val Ala Ala Ser Leu

610 615 620

<210> 22

<211> 362

<212> PRT

<213> Actinoplanes sp.

<400> 22

Met Asn Val Arg Phe Ala Glu Arg Ser Thr Leu Arg Asp Met Arg Ala

1 5 10 15

Tyr Arg Asp Lys Glu Ser Ser Asn Ala Glu Gly Ser Ser Arg Phe Thr

20 25 30

Phe Asp Leu Ser Ser Asn Glu Leu Val Leu Pro Pro Leu Pro Thr Val

35 40 45

Leu Ala Gly Ile Glu Lys Gly Leu Pro Arg Leu Ala Arg Tyr Pro Asp

50 55 60

Pro Thr Ala Arg Asp Leu Thr Glu Asp Ile Ala Gly His Leu Cys Val

65 70 75 80

Ser Pro Asp Glu Val Ala Val Gly Pro Gly Ser Ala Gly Val Leu Gln

85 90 95

Gln Ile Leu Leu Ala Leu Cys Gly Lys Gly Asp Glu Val Val His Gly

100 105 110

Trp Pro Gly Phe Asp Ala Tyr Pro Leu Leu Val Ala Ile Ser Gly Ala

115 120 125

Thr Gly Val His Val Pro Leu Thr Ala Ser Gly Gly His Asp Leu Asp

130 135 140

Glu Ile Arg Thr Arg Val Asn Ala Arg Thr Arg Val Val Ile Leu Cys

145 150 155 160

Ser Pro His Asn Pro Thr Gly Thr Val Ile Asp Gln Asp Glu Leu His

165 170 175

Gly Phe Leu Arg Ser Leu Pro Ala His Val Val Ala Val Leu Asp Glu

180 185 190

Ala Tyr Val Glu Phe Asp Arg Gly Ala Asn Pro Pro Gly Leu Pro Val

195 200 205

Leu Leu Ser Glu His Ser Asn Thr Val Val Leu Arg Thr Phe Ser Lys

210 215 220

Ala Tyr Gly Leu Ala Gly Leu Arg Val Gly Tyr Ala Ala Gly Pro Arg

225 230 235 240

Gln Val Met Ala Thr Val Arg Lys Thr Ala Ile Pro Phe Gly Val Thr

245 250 255

Arg Phe Ala Glu Gln Ala Ala Met Leu Ser Leu Arg Ser Glu Asp Glu

260 265 270

Leu Cys Glu Arg Leu Ala Ala Val Ala Ala Ala Arg Glu Glu Leu Thr

275 280 285

Ala Glu Leu Arg Glu Leu Arg Leu Pro Val Leu Leu Ser Arg Ala Asn

290 295 300

Phe Val Trp Leu Pro Leu Ala Ser Ala Ala Glu Ser Phe Ala Arg Thr

305 310 315 320

Ala Ala Thr Ala Gly Val Lys Val Arg Ala Phe Pro Gly His Gly Val

325 330 335

Arg Ile Ser Val Gly Glu Ala Glu Ala His Arg Thr Leu Leu Ala Ala

340 345 350

Leu Gly Arg Ala Asp Arg Gly Asn Trp Phe

355 360

<210> 23

<211> 318

<212> PRT

<213> Actinoplanes sp.

<400> 23

Met Thr Glu Gln Gly Gly Pro Ser Ile Ala Val Val Gly Ala Gly Gly

1 5 10 15

Val Gly Gly Tyr Phe Gly Gly Leu Leu Ala Ala Ala Gly His Asp Val

20 25 30

Arg Phe Leu Ala Arg Gly Glu Asn Leu Ala Ala Leu Arg Arg Gln Gly

35 40 45

Leu Arg Ile Thr Asn Gly Ser Ser Asp Leu Arg Val Pro Asp Val Arg

50 55 60

Ala Ser Ala Asp Pro Lys Asp Ile Gly Glu Val Asp Phe Val Leu Leu

65 70 75 80

Cys Val Lys Thr Ser Gln Leu Pro Ala Ala Leu Asp Ala Leu Gly Pro

85 90 95

Leu Val Gly Glu His Thr Ala Val Val Thr Val Gln Asn Gly Val Glu

100 105 110

Ala Pro Glu Gln Val Ala Ala Arg Ile Gly Arg Gly Arg Val Leu Pro

115 120 125

Gly Ser Val Arg Val Val Ala Ser Thr Ala Gly Pro Gly Glu Leu Arg

130 135 140

His Val Gly Pro Pro Gly Ala Leu Ala Phe Thr Glu Trp Asp Ser Thr

145 150 155 160

Val Ser Asp Arg Val Ala Arg Leu Arg Glu Val Leu Arg Ala Ala Ser

165 170 175

Val Ser Val Pro Glu Pro Ser Asp Ile Trp Ala Gly Leu Trp Ala Lys

180 185 190

Phe Leu Leu Val Val Pro Ile Gly Ser Leu Gly Ala Ala Thr Gly Gly

195 200 205

Ala Thr Ile Gly Glu Leu Arg Ser Arg Thr Gly Thr Arg Asn Ile Leu

210 215 220

Ile Ala Gly Met Arg Glu Ile Tyr Glu Thr Gly Ile Lys Leu Gly Ile

225 230 235 240

Ala Leu Pro Ala Ala Ala Val Asp Thr Ala Thr Glu Leu Met Asp Gln

245 250 255

Gln Ser Pro Asp Val Thr Ser Ser Leu Gln Arg Asp Ile Leu Ala Gly

260 265 270

Arg Pro Ser Glu Leu Glu Ala Trp Thr Gly Ala Val Val Arg Leu Ala

275 280 285

Arg Gly Ala Gly Leu Thr Ala Pro Val His Glu Met Leu Tyr Glu Leu

290 295 300

Leu Ala Thr Arg Glu Ser Arg Thr Ala Arg Ser Leu Gln Ala

305 310 315

<210> 24

<211> 404

<212> PRT

<213> Actinoplanes sp.

<400> 24

Met Thr Asp Val Ile Pro Thr Glu Phe Phe Thr Glu Pro Gly Ser Asn

1 5 10 15

Pro His Ala Thr Ala Ala Glu Tyr Arg Ser Lys Cys Pro Val His Arg

20 25 30

Ile Asn Val Pro Pro Gly Ala Asp Ala Tyr Ala Val Leu Gly Asn Lys

35 40 45

Val Val Glu Glu Ala Leu Gly Asp Ser Arg Leu Ser Lys Gln Val Glu

50 55 60

Asn Leu Pro Ala Arg Tyr Arg Asp Lys Ala Val Ala Ser Ser Leu Leu

65 70 75 80

Val Val Gly Asn Leu Gly Phe Ala Asp Ala Pro Lys His Thr Arg Leu

85 90 95

Lys Lys Pro Ile Ser Arg Ala Phe Leu Pro Ala Thr Val Ala Gln Leu

100 105 110

Arg Pro Arg Ile Gln Asp Ile Val Asp Asp Leu Ile Asp Thr Phe Pro

115 120 125

Glu Asn Gly Glu Ile Asp Leu Leu Ser Ser Phe Ala Leu Pro Met Pro

130 135 140

Leu Thr Val Ile Cys Glu Tyr Leu Gly Ile Pro Val Ala Asp Arg Pro

145 150 155 160

Leu Phe Leu Glu Trp Ser Tyr Ile Leu Ser Gln Asp Pro Leu Gln His

165 170 175

Asp Glu Ala Glu Leu Lys Ala Ala Ser Glu Glu Phe Thr Asp Tyr Phe

180 185 190

Thr Lys Leu Val Ala Glu Arg Arg Thr Asp Leu Arg Asp Asp Leu Leu

195 200 205

Ser Glu Ile Ile Arg Ala Arg Asp Ala Gly Val Tyr Ser Glu Thr Glu

210 215 220

Leu Leu Ser Thr Leu Leu Leu Leu Ile Ile Ala Gly His Lys Thr Val

225 230 235 240

Ala Asn Met Ile Gly Asn Gly Thr Ala Leu Leu Leu Arg His Pro Gln

245 250 255

Gln Leu Glu Met Leu Arg Ala Thr Pro Glu Leu Ile Pro Ser Ala Ile

260 265 270

Glu Glu Ile Leu Arg Tyr Glu Gly Ser Ala Ala Trp Ala Ser Leu Arg

275 280 285

Val Ala Ala Glu Asp Met Gln Leu Ala Gly Val Asp Ile Pro Lys Gly

290 295 300

Ser Phe Val His Leu Ser Leu Ser Ser Ala Gly Arg Asp Pro Asp Val

305 310 315 320

Tyr Asp Asp Pro Asp Gly Phe Asp Val Thr Arg Ser Pro Asn Arg His

325 330 335

Leu Ser Phe Gly His Gly Pro His Phe Cys Ile Gly Ala Pro Leu Gly

340 345 350

Arg Leu Gln Gly Glu Ile Ala Phe Ser Thr Leu Leu Arg Arg Leu Pro

355 360 365

Arg Phe Glu Leu Ala Val Pro Pro Glu Glu Val Ala Trp Leu Ser Asp

370 375 380

Ser Ser Leu Ser Arg Gly Leu Glu Ala Leu Pro Ile Arg Val Gly Glu

385 390 395 400

Arg Leu Pro Arg

<210> 25

<211> 100

<212> PRT

<213> Actinoplanes sp.

<400> 25

Met Pro Asp Val Lys Leu Pro Ala Ala Phe His Val Leu Thr Gly Gly

1 5 10 15

Arg Arg Gln Leu Pro Val Glu Gly Ala Asn Ile Arg Glu Val Leu Val

20 25 30

Gly Leu Asp Gln Thr Cys Pro Gly Val Leu Glu Arg Leu Met Asp Gln

35 40 45

Glu Gly Ser Val Lys Arg Tyr Val Asn Val Tyr Arg Asn Asp Ser Asp

50 55 60

Ile Arg Ser Leu Asp Gly Leu Glu Thr Lys Val Glu His His Asp Val

65 70 75 80

Ile Trp Ile Val Pro Ala Val Ala Gly Gly Ser Glu Ala Ala Arg Ala

85 90 95

Glu Glu Ser Arg

100

<210> 26

<211> 238

<212> PRT

<213> Actinoplanes sp.

<400> 26

Met Ala Gly Val Lys Asp Ala Gln Tyr Val Thr Ala Ala Thr Asp Asp

1 5 10 15

Gly Leu Gly Gly Thr Ala Asp Ser Ala Ala Leu Leu Asp Asp Leu Pro

20 25 30

Val Thr Val Arg Phe Glu Ile Glu Pro Val Arg Arg Phe Leu Ser Ser

35 40 45

Ala Leu Gly Glu Tyr Gln Lys Cys Leu Asp Ser Arg Asp Ala Asp Gly

50 55 60

Val Pro Ser His Leu Pro Arg Ala Ser Gly Leu Leu Phe Gly Gln Val

65 70 75 80

Gly Gly Ala Glu Ile Val Ile Ser Asp Val Glu Phe Val Pro Asn Val

85 90 95

Arg Asp Ser Asp Glu Ser Val Met Ala Glu Phe Glu Ala Thr Ile Ala

100 105 110

Pro Gln Phe Gly Asp Val Tyr Lys Asn Pro Gly Arg Gly Phe Trp Ser

115 120 125

Asp Glu Gln Gly Val Leu Gln Ala Ile Arg Gln Gln Ser Ala Asn Gly

130 135 140

Leu Glu Leu Leu Gly Ser Ile His Ser His Pro Asn Trp His Glu Ile

145 150 155 160

Gly Pro Pro His Glu Arg Arg Gln Arg Leu Ser Glu His Pro Thr Gln

165 170 175

Met Asp Glu Tyr Leu Phe Arg Gln Ser Cys Trp Pro Val Asn Val Ile

180 185 190

Trp Tyr Val His Glu Ser Ser Gly Gly Ile Ala His Arg Val Ala Ala

195 200 205

Trp Arg Pro Gly Ala Glu Gln Cys Asp Arg Leu Asp Ile Arg Ile Pro

210 215 220

Ala Ala Ile His Glu Gln Phe Glu Val Leu Leu Glu Glu Glu

225 230 235

<210> 27

<211> 496

<212> PRT

<213> Actinoplanes sp.

<400> 27

Met Ser Tyr Asn Gly Thr Ser Pro Arg Pro Pro Ser Ile Ser Ala Thr

1 5 10 15

Met Thr Leu Ile Ala Thr Gly Val Ser Val Leu Ser Tyr Ala Leu Met

20 25 30

Gln Thr Met Val Val Pro Ala Leu His Val Leu Gln Val Gln Leu His

35 40 45

Thr Ala Ser Thr Trp Ser Ala Trp Ile Leu Ser Val Phe Leu Leu Thr

50 55 60

Ser Ala Ala Ser Thr Pro Leu Leu Ser Arg Leu Gly Asp Arg Tyr Ser

65 70 75 80

Lys Arg Lys Val Leu Leu Leu Val Leu Thr Thr Tyr Leu Ile Gly Thr

85 90 95

Val Gly Cys Ala Val Ala Gly Asn Ile Gly Val Leu Ile Ala Cys Arg

100 105 110

Ala Val Gln Gly Val Ser Leu Ala Ala Ile Pro Leu Ser Phe Gly Ile

115 120 125

Leu Arg Asp Val Leu Pro Glu Gln Arg Leu Arg Ser Gly Leu Gly Leu

130 135 140

Val Ser Gly Thr Ile Gly Val Gly Ala Gly Ile Gly Leu Val Val Gly

145 150 155 160

Gly Leu Val Val Asp His Gln Ser Trp Arg Trp Leu Phe Ala Val Ala

165 170 175

Ala Val Leu Ile Leu Gly Ala Ile Gly Leu Val Ala Lys Tyr Val Pro

180 185 190

Asp Gln Arg Gly Glu Ala Gly Glu Pro Val Asp Val Pro Gly Ala Val

195 200 205

Leu Leu Ala Leu Val Leu Val Ala Leu Leu Leu Ala Leu Thr Lys Gly

210 215 220

Thr Ser Trp Gly Trp Ala Ser Thr Gly Thr Leu Ala Leu Phe Gly Ala

225 230 235 240

Ser Ala Val Leu Leu Gly Leu Leu Val Val Val Glu Arg Lys Ser Pro

245 250 255

Ala Pro Leu Ile Asp Pro Ala Val Val Ala Gly Arg Ser Phe Val Ser

260 265 270

Val His Gly Ala Ala Phe Val Phe Gly Val Val Ser Phe Val Phe Tyr

275 280 285

Val Leu Leu Pro Thr Tyr Ala Gln Thr Ala Ala Asp Gln Arg Leu Pro

290 295 300

Gly Gly Gly Thr Ile Gly Tyr Gly Leu Gly Ala Asp Val Thr Met Ala

305 310 315 320

Gly Leu Leu Leu Leu Pro Gly Ser Leu Val Leu Leu Pro Ala Gly Pro

325 330 335

Leu Ala Gly Leu Leu Gln Arg Leu Thr Ser Val Arg Ala Thr Leu Ala

340 345 350

Ser Gly Phe Ala Val Met Ala Val Gly Ala Ile Ser Leu Trp Ala Trp

355 360 365

Asn Ala Asn Gly Trp Gln Val Ala Val Gly Tyr Leu Val Val Gly Leu

370 375 380

Gly Ser Gly Leu Val Leu Ser Gly Leu Pro Ser Val Ile Ser Asp Leu

385 390 395 400

Thr Glu Ala Arg Arg Thr Ala Thr Ala Asn Gly Val Asn Thr Val Val

405 410 415

Arg Thr Ala Gly Gly Val Val Gly Ser Gln Leu Ala Val Ala Leu Leu

420 425 430

Ala Ala Trp His Ile Ser Gly Ser Asp Thr Pro Ala Arg Asp Gly Phe

435 440 445

Thr Thr Ala Phe Trp Ile Ala Ala Ala Val Ala Ala Ala Gly Gly Leu

450 455 460

Leu Cys Trp Val Gly Ile Lys Thr Ser Thr Leu Arg Gly Pro Arg Met

465 470 475 480

Pro Gly Val Thr Asp Leu Pro Arg Gln Ser Ala Gly Gly Val Arg Pro

485 490 495

<210> 28

<211> 313

<212> PRT

<213> Actinoplanes sp.

<400> 28

Met Glu Leu Asp Leu Arg His Leu Arg Tyr Phe Val Ala Val Ala Glu

1 5 10 15

Glu Gly Gly Phe Thr Arg Ala Ala Ala Arg Leu His Met Thr Gln Pro

20 25 30

Pro Leu Ser Val Ala Ile Arg Gln Leu Glu Arg Glu Leu Gly Leu Gln

35 40 45

Leu Leu Asp Arg Thr Gly Asn Arg Val Glu Leu Thr Ser Val Gly Arg

50 55 60

Asp Phe Leu Thr His Ala Arg Asn Leu Leu Gln Gln Trp Gln Val Thr

65 70 75 80

Val Glu Arg Met Arg Gln Ala Gly Ser Gln Asp Val Glu Arg Leu Val

85 90 95

Val Ala Phe Arg Pro Ala Val Ser Arg Pro Leu Ala His Arg Thr Ile

100 105 110

Glu Leu Ile Arg Glu Lys His Pro Glu Tyr Gln Val Val Pro Arg Tyr

115 120 125

Val Pro Trp Thr Glu Gln Thr Ala Cys Leu Glu Ala Gly Asp Ala Asp

130 135 140

Val Ser Phe Val Leu Glu Pro Ala Asp Tyr Val Gly Leu Glu Arg Ala

145 150 155 160

Thr Val Ala Leu Leu Pro Arg Val Val Cys Leu Pro Ser Ala His Glu

165 170 175

Leu Ala Ser Arg Asp Ser Val Ser Ile Asp Asp Leu Ser Glu Val Pro

180 185 190

Ile Ile Arg Pro Thr Gly Gly Ser Pro Glu Trp Ser Asp Phe Trp Gly

195 200 205

Gly Glu Val Cys Pro Gly Lys Arg Thr Trp Lys Glu Pro Pro Thr Ala

210 215 220

Thr Arg Leu Asp Glu Ala Ile Asp Leu Val Ala Leu Glu Asn Ala Ala

225 230 235 240

Ala Leu Val Pro Val Ser Val Met Ala Val Gln His Arg Gln Asp Val

245 250 255

Val Phe Ile Pro Val Thr Asp Val Pro Ala Ala Arg Leu Ser Leu Ala

260 265 270

Trp Arg Glu Gly Ser Asp Ser Glu Leu Val Arg Leu Ala Val Arg Cys

275 280 285

Ala Gln Ala Ala Ala Gln Asp Pro Ala Val Arg Thr Leu Phe Gly Glu

290 295 300

Pro Arg Pro Thr Gly Thr Ala Pro Ala

305 310

<210> 29

<211> 300

<212> PRT

<213> Actinoplanes sp.

<400> 29

Met Arg Arg Trp Ala Ala Gln Ser Gly Pro Glu Asp Leu Tyr Phe Val

1 5 10 15

Ser Asn Leu His Ala Met Thr Thr Lys His Asp Pro Glu Arg Leu Gln

20 25 30

Glu Leu Thr Asp His Gln Leu Ala Leu Leu Ile Ala Ala Gly Val Pro

35 40 45

Gln Glu Arg Leu Phe Val Gln Ser Asp Leu Ile Gln Glu His Met Ala

50 55 60

Leu Thr Trp Leu Leu Glu Cys Thr Cys Thr Phe Gly Glu Ala Arg Arg

65 70 75 80

Met Val Gln Phe Lys Glu Lys Ser Gln Gly Ser Asn Ser Val Arg Leu

85 90 95

Gly Leu Leu Thr Tyr Pro Val Leu Met Ala Ala Asp Ile Leu Leu His

100 105 110

Gly Ala Ser Glu Val Pro Val Gly His Asp Gln Asn Gln His Val Glu

115 120 125

Leu Ala Arg Thr Leu Ala Arg Arg Phe Asn Thr Asp Tyr Gly Glu Val

130 135 140

Phe Thr Val Pro Gln Ala Val Leu Pro Val Ala Ala Ala Arg Val Arg

145 150 155 160

Asp Leu Ala Ala Pro Thr Arg Lys Met Ser Lys Ser Ser Ser Asp Gly

165 170 175

Ser Gly Ile Val Tyr Val Leu Asp Ser Pro Glu Ala Val Arg Arg Lys

180 185 190

Phe Gln Arg Ala Val Thr Asp Gly Glu Asn Thr Val Arg Tyr Ala Pro

195 200 205

Asp Glu Gln Pro Gly Val Ala Asn Leu Leu Glu Ile Arg Ala Ala Cys

210 215 220

Thr Asp Thr Leu Pro Ser Asp Ala Ala Lys Gly Ile Asp Ser Tyr Arg

225 230 235 240

Asp Leu Lys Glu Ala Ala Ala Glu Ala Val Ile Ser Leu Ile Ala Pro

245 250 255

Val Arg Glu Arg Ala Leu Gln Leu Leu Glu Glu Arg Ser Glu Leu Ala

260 265 270

Lys Ile Arg Ala Glu Gly Ala Asp Arg Ala Arg Ala Arg Ser Arg Asp

275 280 285

Arg Leu Asp Arg Ala Leu Ser Leu Ala Gly Leu Lys

290 295 300

<210> 30

<211> 264

<212> PRT

<213> Actinoplanes sp.

<400> 30

Met Ala Asp Asp Pro Leu Val Ile Gly Gly Thr Ser Tyr Ser Ser Arg

1 5 10 15

Leu Ile Met Gly Thr Gly Gly Ala Pro Ser Leu Asp Val Leu Glu Arg

20 25 30

Ser Leu Val Ala Ser Gly Thr Glu Leu Thr Thr Val Ala Met Arg Arg

35 40 45

Val Asp Pro Ser Val Lys Gly Ser Val Leu Ser Val Leu Asp Arg Leu

50 55 60

Gly Ile Gln Val Leu Pro Asn Thr Ala Gly Cys Phe Thr Ala Gly Glu

65 70 75 80

Ala Val Leu Thr Ala Arg Leu Ala Arg Glu Ala Leu Gly Thr Asp Leu

85 90 95

Val Lys Leu Glu Val Ile Ala Asp Glu Arg Thr Leu Leu Pro Asp Pro

100 105 110

Ile Glu Thr Leu Glu Ala Ala Glu Thr Leu Val Asp Asp Gly Phe Thr

115 120 125

Val Leu Pro Tyr Thr Asn Asp Asp Pro Val Leu Ala Arg Lys Leu Gln

130 135 140

Asp Val Gly Cys Ala Ala Ile Met Pro Leu Gly Ser Pro Ile Gly Ser

145 150 155 160

Gly Leu Gly Ile Arg Asn Pro His Asn Phe Gln Leu Ile Val Glu His

165 170 175

Ala Cys Val Pro Val Ile Leu Asp Ala Gly Ala Gly Thr Ala Ser Asp

180 185 190

Ala Ala Leu Ala Met Glu Leu Gly Cys Ala Ala Val Met Leu Ala Ser

195 200 205

Ala Val Thr Arg Ala Gln Glu Pro Val Leu Met Ala Glu Gly Met Arg

210 215 220

His Ala Val Glu Ala Gly Arg Leu Ala His Arg Ala Gly Arg Ile Pro

225 230 235 240

Arg Arg His Phe Ala Glu Ala Ser Ser Pro Thr Glu Gly Met Ala Arg

245 250 255

Leu Asp Pro Glu Arg Pro Ala Phe

260

<210> 31

<211> 392

<212> PRT

<213> Actinoplanes sp.

<400> 31

Met Ser Leu Pro Pro Leu Val Glu Pro Ala Ala Glu Leu Thr Val Asp

1 5 10 15

Glu Val Arg Arg Tyr Ser Arg His Leu Ile Ile Pro Asp Val Gly Met

20 25 30

Asp Gly Gln Lys Arg Leu Lys Asn Ala Lys Val Leu Cys Val Gly Ala

35 40 45

Gly Gly Leu Gly Ser Pro Ala Leu Met Tyr Leu Ala Ala Ala Gly Val

50 55 60

Gly Thr Leu Gly Ile Val Glu Phe Asp Glu Val Asp Glu Ser Asn Leu

65 70 75 80

Gln Arg Gln Ile Ile His Ser Gln Ala Asp Ile Gly Arg Ser Lys Ala

85 90 95

Glu Ser Ala Lys Asp Ser Val Leu Gly Ile Asn Pro Tyr Val Asn Val

100 105 110

Ile Leu His Glu Glu Arg Leu Glu Ala Glu Asn Val Met Asp Ile Phe

115 120 125

Ser Gln Tyr Asp Leu Ile Val Asp Gly Thr Asp Asn Phe Ala Thr Arg

130 135 140

Tyr Leu Val Asn Asp Ala Cys Val Leu Leu Asn Lys Pro Tyr Val Trp

145 150 155 160

Gly Ser Ile Tyr Arg Phe Asp Gly Gln Ala Ser Val Phe Trp Ser Glu

165 170 175

His Gly Pro Cys Tyr Arg Cys Leu Tyr Pro Glu Pro Pro Pro Pro Gly

180 185 190

Met Val Pro Ser Cys Ala Glu Gly Gly Val Leu Gly Val Leu Cys Ala

195 200 205

Ser Ile Gly Ser Ile Gln Val Asn Glu Ala Ile Lys Leu Leu Ala Gly

210 215 220

Ile Gly Asp Pro Leu Val Gly Arg Leu Met Ile Tyr Asp Ala Leu Glu

225 230 235 240

Met Gln Tyr Arg Gln Val Lys Val Arg Lys Asp Pro Asn Cys Ala Val

245 250 255

Cys Gly Glu Asn Pro Thr Val Thr Glu Leu Ile Asp Tyr Glu Ala Phe

260 265 270

Cys Gly Val Val Ser Glu Glu Ala Gln Glu Ala Ala Leu Gly Ser Thr

275 280 285

Ile Thr Pro Lys Gln Leu Lys Glu Trp Ile Asp Asp Gly Glu Asn Ile

290 295 300

Asp Ile Ile Asp Val Arg Glu Gln Asn Glu Tyr Glu Ile Val Ser Ile

305 310 315 320

Pro Gly Ala Arg Leu Ile Pro Lys Asn Glu Phe Leu Met Gly Gly Ala

325 330 335

Leu Gln Asp Leu Pro Gln Asp Lys Lys Ile Val Leu His Cys Lys Thr

340 345 350

Gly Val Arg Ser Ala Glu Val Leu Ala Val Leu Lys Ser Ala Gly Phe

355 360 365

Ala Asp Ala Val His Val Gly Gly Gly Val Ile Gly Trp Val Asn Gln

370 375 380

Ile Glu Pro Ser Lys Pro Val Tyr

385 390

<210> 32

<211> 424

<212> PRT

<213> Actinoplanes sp.

<400> 32

Met Ser Met Glu Asn Pro Arg Gly Arg Arg His Gly Leu Arg Leu Ala

1 5 10 15

Ala Ala Ala Val Ala Thr Ala Ser Leu Met Ser Gly Val Leu Leu Ser

20 25 30

Thr Ala Asp Glu Ser Val Ala Ala Pro Pro Ser Thr Tyr Glu Val Lys

35 40 45

Gly Val Asp Thr Ser His His Asn His Asp Ala Thr Gly Lys Pro Ile

50 55 60

Asp Trp Lys Arg Val Ala Gln Ser Asn Ser Phe Ala Phe Leu Lys Ala

65 70 75 80

Thr Gln Gly Thr Gly Tyr Lys Asp Pro Trp Phe Ala Arg Asp Phe Lys

85 90 95

Asp Ala Ser Gly Thr Ser Leu Leu Arg Ala Pro Tyr His Phe Phe Asp

100 105 110

Pro Lys Ser Thr Thr Asp Gly Gly Ala Gln Ala Asp His Phe Ile Arg

115 120 125

Ala Ala Arg Ser Ala Gly Tyr Thr Gly Lys Arg Ala Gly Glu Leu Pro

130 135 140

Pro Val Leu Asp Val Glu Gly Thr Trp Val Asn Gly Lys Glu Val Cys

145 150 155 160

Pro Lys Ala Leu Arg Ala Asp Gln Leu Thr Ala Phe Leu Asn Arg Val

165 170 175

Glu Glu Ala Phe Lys Val Thr Pro Ile Val Tyr Thr Arg Ala Ser Phe

180 185 190

Val Asn Gly Cys Met Ala Gly Lys Gly Gln Val Phe Lys Asp His Pro

195 200 205

Leu Trp Leu Ala Arg Tyr Glu Ser Gly Ser Lys Glu Pro Gln Asp Val

210 215 220

Pro Gly Ala Gly Ala Trp Ser Leu Trp Gln Tyr Thr Glu Ser Glu Ala

225 230 235 240

Ile Pro Gly Leu Pro Gly Lys Asn Gly Ala Ala Gly Lys Gly Asp Arg

245 250 255

Asn Val Tyr Arg Gly Ser Leu Asp Gln Leu Arg Ala Leu Ala Lys Gly

260 265 270

Gly Gly Ala Pro Gln Pro Gln Pro Gly Thr Ser Trp Pro Thr Val Lys

275 280 285

Ala Gly Asp Lys Gly Val Asp Val Ala Thr Val Gln Leu Leu Leu Gly

290 295 300

Ala His Gly Tyr Ala Thr Thr Ala Asp Gly Val Phe Gly Thr Gly Thr

305 310 315 320

Ala Ala Lys Val Gln Ala Phe Gln Lys Ala Glu Gly Leu Ala Ala Asp

325 330 335

Gly Met Val Gly Pro Ala Thr Trp Ala Lys Leu Ile Ala Thr Val Lys

340 345 350

Ser Gly Ser Lys Gly Thr Asp Val Thr Ala Leu Gln Arg Gln Leu Ala

355 360 365

Asp Asn Gly Tyr Asp Val Thr Ala Asp Gly Val Phe Gly Pro Ala Thr

370 375 380

Thr Ser Lys Leu Thr Ala Phe Gln Lys Ala Lys Gly Leu Thr Ser Asp

385 390 395 400

Gly Ile Ala Gly Pro Ala Thr Trp Ala Lys Leu Val Ser Gly Gly Thr

405 410 415

Ala Gly Ala Thr Ala Thr Thr Ser

420

<210> 33

<211> 331

<212> PRT

<213> Actinoplanes sp.

<400> 33

Met Gly Val Glu Arg Asp Glu Pro Thr Arg Ser Ala Arg Arg Glu Leu

1 5 10 15

Ala Leu Leu Leu Arg Ser Trp Trp Glu Ala His Pro Asp Lys Ile Thr

20 25 30

Gln Glu Ala Leu Ala Arg Arg Ile Thr Glu Arg Gly Val Arg Ile Ser

35 40 45

Gln Glu Met Leu Ser Arg Tyr Leu Asn Arg Ser Arg Pro Thr Thr Ala

50 55 60

Arg Pro Asp Val Ile Arg Thr Met His Glu Val Leu Arg Arg Ala Pro

65 70 75 80

Glu Glu Leu Asp Val Ala Leu Glu Leu His Ala Arg Ala Thr Ala Pro

85 90 95

Gln Thr Pro Pro Ala Glu Gly Ala Ala Thr Ser Gln Pro Ala Gly Asp

100 105 110

Ala Gly Thr Ala Ala Pro Lys Gly Val Glu Pro Thr Ser Ala Ala Pro

115 120 125

Leu Leu Thr Arg Thr Pro His Thr Pro Arg Pro Ala Ser Arg Lys Lys

130 135 140

Trp Pro Trp Ile Ala Val Val Ala Ala Ala Val Val Gly Ala Ser Gly

145 150 155 160

Leu Thr Ala Phe Met Thr Leu Gly Asp Gln Arg Gln Asn Thr Pro Arg

165 170 175

Gly His Gly Ala Thr Pro Ser Ala Ser Pro Thr Ala Leu Val Ser Pro

180 185 190

Thr Ala Gln Gly Ser Pro Ala Gly Thr His Pro Pro Ala Glu Cys Arg

195 200 205

Asp Glu Ser Cys Phe Gly Ile Asp Ala Lys Tyr Ala Ile Cys Gln Asp

210 215 220

Asp Ala Ala Thr Tyr Tyr Thr Gly Arg Ala His Gly Val Leu Val Glu

225 230 235 240

Leu Arg Phe Ser Pro Ala Cys Gln Ala Ala Trp Ala Lys Met Ser Gly

245 250 255

Thr Ser Gln Gly Asp Val Val Arg Val Thr Asn Asn Ala Gly Arg Ser

260 265 270

Arg His Tyr Thr Gln Gln Trp Gly Arg Asp Ala His Thr Thr Met Val

275 280 285

Glu Ala Val Ser Pro Asp Asp Ala Lys Ala Cys Ala Arg Thr Pro Arg

290 295 300

Gly Glu Val Cys Ala Thr Lys Ala Val Ala Ser Ala Pro Arg Asp Ala

305 310 315 320

Ala Pro Gly Glu Arg Ala Ala Pro Gly Gly Arg

325 330

<210> 34

<211> 346

<212> PRT

<213> Actinoplanes sp.

<400> 34

Met Pro Ala Arg Thr Thr Arg Thr Ala His Thr Thr Arg Thr Gly Arg

1 5 10 15

Leu Ala Val Val Ala Leu Ala Ala Leu Thr Cys Ala Gly Leu Val Thr

20 25 30

Gly Thr Ala Ala Thr Ala Thr Thr Pro Asp Ser Leu Pro Thr Ala Lys

35 40 45

Arg Ala Ala Ala Pro Asp Ala Ala Ala Val Ser Trp Pro Thr Leu Lys

50 55 60

Ala Gly Ala Arg Gly Thr Glu Val Thr Ala Leu Gln His Leu Leu Ile

65 70 75 80

Ala Arg Gly Gln Ser Val Ala Val Asp Gly Glu Phe Gly Pro Ala Thr

85 90 95

Thr Thr Ala Val Lys Ala Phe Gln Lys Ala Asp Gly Leu Thr Ala Asp

100 105 110

Gly Ile Val Gly Pro Ala Thr Trp Ala Lys Leu Val Pro Thr Leu Arg

115 120 125

Gln Gly Ala Gln Gly Ala Ala Val Lys Ala Ala Gln Thr Leu Leu Lys

130 135 140

Thr Arg Gly Gln Ser Val Ala Val Asp Gly Glu Phe Gly Ser Ala Thr

145 150 155 160

Thr Ser Ala Val Lys Ala Phe Gln Lys Ala Lys Gly Leu Ser Ala Asp

165 170 175

Gly Val Val Gly Thr Gln Ser Trp Ser Ala Leu Leu Thr Ser Asp Ser

180 185 190

Gly Ala Pro Ser Gly Asn Arg Ala Ala Phe Ala Gln Gln Ile Leu Asn

195 200 205

Thr Ser Gly Ile Glu Leu Ala Thr Val His Pro Gly Gly Thr His Ala

210 215 220

Gly Ser Thr Ala Arg Gln Asn Ile Ile Asp Thr Ala Asn Gly Lys Gly

225 230 235 240

Ala Leu Thr Ser Pro Trp Ser Asp Lys Pro Asn Gln Arg Val Ala Leu

245 250 255

Asp Thr Arg Met Leu Asn Gly Leu Leu Lys Leu Leu Ser Gln Asp Gly

260 265 270

Tyr Arg Ile Ser Val Ser Glu Ile Val Gly Gly Asp His Ser Thr Asn

275 280 285

Ser Arg His Tyr Ala Gly Leu Gly Phe Asp Ile Asn Tyr Ile Asn Gly

290 295 300

Arg His Val Gly Glu Ser Ala Pro His Gln Gly Leu Met Ala Ala Cys

305 310 315 320

Arg Lys Leu Gly Ala Thr Glu Val Leu Gly Pro Gly Asp Ala Gly His

325 330 335

Ser Arg His Val His Cys Gly Trp Pro Arg

340 345

<210> 35

<211> 240

<212> PRT

<213> Actinoplanes sp.

<400> 35

Met Leu Asp Ile Asp Glu Leu Lys Ala Arg Asp Ser Asp Glu Gly Arg

1 5 10 15

Val Pro Ala Gly Gly Arg Pro Ala Thr Glu Thr Leu Thr Leu Gly Leu

20 25 30

Asp Arg Ala Glu Leu Pro Val Ala Thr Glu Leu Ala Ala Leu Leu His

35 40 45

Arg Val Pro Val Ala Gly Val Arg Leu Pro Glu Pro Ala Asp Phe Ser

50 55 60

Ala Leu Pro Ser His Val Ile Val Arg Ile Ile Ala Leu Ile Arg Glu

65 70 75 80

Cys Ser Ser Ile Gly Thr Arg Val Thr Trp Ser Leu Thr Leu Gly Ala

85 90 95

Glu Gln Leu Asp Leu Val Pro Arg Leu Asp His Leu Pro Ala Pro Asp

100 105 110

Ser Ile Thr Val Leu Glu Thr Gly His Pro Ser Val Gly Glu Trp Arg

115 120 125

Ser Ser Ser Asn Phe Gly Leu Leu Tyr Phe Arg Lys Gly Pro Lys Phe

130 135 140

Leu Ser Val Val Asp Gln Arg Pro Glu Ser Ser Arg Glu Ile Ile Val

145 150 155 160

Asp Asp Pro Thr Gln Met Ala Val Phe Leu Leu Gly Leu Glu Gly Cys

165 170 175

Ala Trp Ala Glu Val Thr Arg Asn Ser Gln Phe Ala Ala Ala Ala Arg

180 185 190

Asp Leu Val Asn Lys Gly Leu Val Met Arg Val Gly Asp His Cys Val

195 200 205

Thr Leu Pro Val His Met Arg Ser Trp Pro Leu Gly Ala Ala Leu Leu

210 215 220

Gly Gly Thr Leu Ala Ala Ala Gly Lys Lys Ser Asp Gly Ala Thr Glu

225 230 235 240

<210> 36

<211> 846

<212> PRT

<213> Actinoplanes sp.

<400> 36

Met Ser Ala Val Leu Gly Val Leu Leu Ala Ile Ser Leu Ala Thr Ala

1 5 10 15

Pro Ala His Ala Ala Val Arg Ser Ala Ala Ala Val Asp Val Cys Arg

20 25 30

Ser Ala Ala Leu Ser Lys Ala Arg Val Ser Thr Trp Val Arg Leu Glu

35 40 45

His Arg Asp Gly Thr Tyr Ser Arg Ile Arg Ser Glu Leu Ser Val Glu

50 55 60

Val Pro Glu Asp Trp Pro Leu Ala Lys Asp Leu Leu Leu Ser Glu Asp

65 70 75 80

Ser Arg Arg Tyr Val Ala Ala Met Ser Cys Leu Thr Arg Thr Asp Arg

85 90 95

Gly Arg Gln Arg Arg Trp Ser Glu Trp Arg Ser Ser Arg Pro Thr Val

100 105 110

Ala Ser Thr Lys Ser Gly Gly Val Lys Val Val Asp Arg Thr His Ser

115 120 125

Trp Val Asn Val Tyr Arg Ala His Ile Asp Val Gly Thr Trp Arg Val

130 135 140

Arg Ala Gly Ala Glu Arg Trp Thr Val Gln Leu Gln Ala Pro Ser Ala

145 150 155 160

Leu Asn Ala Ala Arg Trp Asp Glu Ile Arg Val Glu Pro Gly Ala Pro

165 170 175

Gly Ala Glu Ser Ala Thr Pro Arg Pro Asp Glu Gly Arg Gly Ala Thr

180 185 190

Ala Leu Val Trp His Pro Gln Asn His Arg Glu Lys Ala Ala Ala Pro

195 200 205

Ala Val Ser Val Ala Leu Lys Pro Ser Trp Gln Arg Ser Trp Ala Ala

210 215 220

Gln Asn Asp Arg Leu Val Ala Val Ala Leu Asp Arg Gly Gly Trp Leu

225 230 235 240

Leu Trp Asp Ala Thr Ser Ala Ala Leu Leu Leu Tyr Ala Thr Val Leu

245 250 255

Tyr Arg Arg Arg Ser Ala Pro Pro Thr Gln Ala Gln Glu Arg Thr Leu

260 265 270

Arg Asn Leu Ser Leu Trp Ala Lys Ala Leu Val Val Leu Val Ala Leu

275 280 285

Thr Ser Met Asp Asp Val Leu Ile Arg Tyr Val Gln Arg Arg Gly Asp

290 295 300

Gly Leu Leu Leu Asp Glu Gln Ile Pro Arg Gly Asn Ala Phe Ala Leu

305 310 315 320

Ala Ala Val Ile Val Leu Phe Cys Val Gly Arg Pro Arg Arg Arg Ile

325 330 335

Trp Ala Ala Ala Ala Val Leu Ala Val Pro Thr Val Ala Ala Leu Pro

340 345 350

Gln Trp Phe Glu Leu Ser Pro Gln Arg Phe Val Ser Asp Asp Glu Trp

355 360 365

Ala Val Thr Leu Ala Ala Gln Gly Val Ala Ala Cys Cys Met Leu Ala

370 375 380

Leu Leu Gly Leu Gly Phe Val Thr Ala Ala Trp Arg Leu Ala Val Asp

385 390 395 400

Gly Asp Leu Leu Pro Met Ser Arg Arg His Pro Gly His Ala Arg Val

405 410 415

Leu Arg Leu Arg Ile Ala Gly Pro Val Ile Leu Val Cys Thr Ala Ala

420 425 430

Val Ala Ile Cys Phe Ala Leu Ala Gln Glu Arg Asn Trp Gln Arg Ala

435 440 445

Thr Trp Leu Ser Asp Arg Ser Asp Pro Ala Tyr Ala Thr Gly Gln Trp

450 455 460

Ser Asp Arg Val Trp Glu Ala Val Trp Ser Val Ala Asn Gly Gln Asp

465 470 475 480

Trp Leu Ser Trp Gln Ala Trp Leu Leu Thr Gly Val Ala Val Leu Ala

485 490 495

Val Leu Arg Thr Trp Arg Ala Pro Ala Ser Val Ser Pro Leu Asp Asp

500 505 510

Pro Ala Asp Arg Leu Leu Phe Leu Ala Phe Phe Ala Ile Val Ala Ala

515 520 525

Ala Ser Gly Gly Tyr Phe Leu Gly Asn Glu Val Leu Thr Gly Leu Trp

530 535 540

Ile Pro Leu Ser Met Leu Ala Leu Tyr Trp Val Val Val Pro Phe Thr

545 550 555 560

His Arg Ser Val Leu Ala Gln Pro Phe Glu Arg Ser Gly Arg Pro Leu

565 570 575

Ala Asp Ser Ala Gly Pro Gly Ala Arg Thr Val Leu Leu Ala Lys Ala

580 585 590

Arg Ser Tyr Arg Glu Thr His Ala Glu Leu Arg Arg Leu Asp Gln Gly

595 600 605

Leu Phe Gly Asp Val Pro Pro Lys Arg Ser Asp Leu Glu Gln Glu Leu

610 615 620

Ser Asp Leu His Asn Trp Pro Thr Ala Gly Gly Ser Asp Arg Leu Pro

625 630 635 640

Ala Lys Val Ser Val Val Asp Gly Ala Leu Ala Leu Gly Pro Arg Asp

645 650 655

Thr Trp Trp Ala Asn Gly Ser Arg Cys Ala Arg Leu Ala Leu Val Pro

660 665 670

Ala Val Pro Ala Ala Leu Leu Leu Ala Trp Val Trp Lys Val Lys Gly

675 680 685

Glu Ala Trp His Ala Thr Leu His Glu Gln Phe Gly Leu Pro Asp Val

690 695 700

Leu Leu Leu Phe Val Gly Glu Met Val Met Phe Thr Ser Ser Ala Phe

705 710 715 720

Val Leu Gly Ala Leu Trp Arg His Leu Pro Gly Gln Arg Gly Ala Ala

725 730 735

Lys Ala Leu Pro Val Thr Leu Ala Phe Ala Leu Pro Ile Gly Leu Asp

740 745 750

Ala Leu Val Tyr Arg Phe Thr Gly Glu Ser Thr Ala Asn Leu Ala Leu

755 760 765

Ala Val Ser Ala Met Leu Phe Val Leu Thr Val Thr Ser Ile Ala Leu

770 775 780

Asp Phe Asp Thr Phe Arg Gly Glu Arg Arg Tyr Trp Gln Ser Arg Leu

785 790 795 800

Gly Leu Leu Leu Ser Ile Tyr Gln Met Arg Tyr Tyr Ser Leu Gln Ala

805 810 815

Ala Tyr Leu Ile Ala Gln Val Val Ala Met Ile Thr Ile Trp Glu Phe

820 825 830

Phe Ala Glu Pro Asp Val Val Pro Lys Pro Ser Asp Ser Lys

835 840 845

<210> 37

<211> 548

<212> PRT

<213> Actinoplanes sp.

<400> 37

Met Leu Thr Leu Thr Val Pro Ala Met Ile Thr Phe Gly Val Phe Leu

1 5 10 15

Ile Ala Met Val Met Ile Gly Val Met Thr Gln Lys Glu Thr Ala Thr

20 25 30

Phe Ala Asp Phe Thr Val Gly Gly Arg Arg Leu Thr Ala Pro Met Ala

35 40 45

Ala Leu Ser Ala Gly Ala Ser Asp Met Ser Gly Trp Leu Phe Leu Gly

50 55 60

Leu Pro Gly Ala Val Tyr Met Ala Gly Ile Gly Ala Thr Trp Ile Ala

65 70 75 80

Val Gly Leu Ile Val Gly Thr Tyr Leu Asn Trp Arg Phe Val Ala Pro

85 90 95

Arg Leu Arg Thr Tyr Thr Glu Leu Ala Gly Asn Ser Val Thr Leu Pro

100 105 110

Ser Tyr Leu Glu Glu Arg Phe Glu Asp Arg Ser Arg Met Leu Arg Leu

115 120 125

Leu Ser Ala Ile Val Thr Val Leu Phe Phe Thr Val Tyr Val Ala Ser

130 135 140

Gly Leu Val Ala Gly Gly Leu Leu Phe Asn Glu Ile Phe Gly Ala Asp

145 150 155 160

Phe Glu Phe Gly Leu Thr Val Phe Ala Val Val Ile Val Ala Tyr Thr

165 170 175

Ile Leu Gly Gly Phe Arg Ala Val Ser Ile Thr His Ser Ile Gln Gly

180 185 190

Thr Leu Met Phe Leu Ala Ala Leu Val Leu Pro Ala Leu Gly Leu Trp

195 200 205

Arg Leu Gly Gly Phe Gly Ala Leu His Asp Ala Leu Ser Asp Lys Thr

210 215 220

Pro Ala Leu Leu Asp Pro Val Ala Glu Ala Ser Phe Ala Gly Asn Thr

225 230 235 240

Trp Ser Ala Gly Glu Pro Leu Gly Ala Ile Ala Met Ile Ser Leu Leu

245 250 255

Ala Trp Gly Leu Gly Tyr Phe Gly Gln Pro His Ile Leu Ile Arg Phe

260 265 270

Met Gly Ile Arg Ser Thr Lys Asp Ile Pro Leu Ala Arg Arg Leu Gly

275 280 285

Val Gly Trp Val Val Val Val Leu Gly Gly Ser Ser Leu Ile Gly Leu

290 295 300

Ala Gly Ile Ala Val Leu Asp Glu Pro Leu Asp Asn Pro Glu Thr Val

305 310 315 320

Tyr Ile Glu Leu Ser Thr His Leu Val Asn Pro Trp Ile Ala Gly Ile

325 330 335

Leu Leu Val Ala Val Leu Ala Ala Ile Lys Ser Thr Val Asp Ser Gln

340 345 350

Leu Leu Val Ser Ala Thr Ser Leu Thr Glu Asp Phe Tyr Arg Ala Phe

355 360 365

Leu Asn Arg Arg Ala Ser Asp Thr Leu Leu Leu Met Val Gly Arg Leu

370 375 380

Ser Val Val Ala Val Ala Leu Val Ala Tyr Ala Ile Ala Leu Ser Gly

385 390 395 400

Gly Ala Val Leu Asp Ile Val Ala Tyr Ala Trp Ala Gly Phe Gly Ala

405 410 415

Ala Phe Gly Pro Val Ile Ile Leu Ser Leu Phe Trp Pro Arg Met Thr

420 425 430

Ala Ala Gly Ala Met Ala Gly Met Val Thr Gly Ala Leu Thr Val Phe

435 440 445

Leu Trp Lys Tyr Ile Asp Pro Leu Leu Gly Pro Leu Glu Ser Gly Val

450 455 460

Tyr Glu Met Val Pro Gly Val Leu Ala Ala Thr Ala Ala Ala Leu Leu

465 470 475 480

Phe Gly Lys Tyr Val Gly Arg Pro Pro Ala Arg Ala Trp Ile Gly Ser

485 490 495

Met Glu His Ala Thr Thr Glu Leu Pro Thr Pro Glu His Ser Thr Thr

500 505 510

Glu His Pro Thr Met Glu Gln Gly Ser Thr Asp Tyr Gly Thr His Gln

515 520 525

Gly Ser Thr Asp Tyr Gly Thr Tyr Gln Gly Leu Pro Gln Ser Gln Asp

530 535 540

Gln Trp Arg Pro

545

<210> 38

<211> 79

<212> PRT

<213> Actinoplanes sp.

<400> 38

Met Val Arg Arg Thr Gly Gly Arg Arg Gly Asp Asn Gly Ala Gln Glu

1 5 10 15

Pro Thr Ser His Gly Asp Asp His Ala Thr Gly Asn Gly His Thr Ser

20 25 30

Thr Arg Ser Ser Asn Cys Thr Ala Gln Pro Lys Cys Thr Ala His Thr

35 40 45

Asn Pro Val Asn Arg Ser Ala Thr Thr Glu Gly His Ala Gly Ala Ala

50 55 60

Ala Ser Pro Gly Ile Ala Gln Gly Pro Trp Gly Glu Ala Val Gly

65 70 75

<210> 39

<211> 651

<212> PRT

<213> Actinoplanes sp.

<400> 39

Met Thr Val Asp Thr Pro Thr Arg Ala Ala Lys Glu Lys Leu Ala Gly

1 5 10 15

Leu Gly Leu Asp Leu Thr Glu His Ala Gly His Gly Phe Val Glu Val

20 25 30

Val Leu His Ser Pro Ala Asp Ala Leu Ala Leu Gln Val Gly Gly Phe

35 40 45

Ser Trp Lys Val Arg Val Pro Asp Leu Val Gln Arg Glu Ser Asp Val

50 55 60

Asn Ala Ala Asn Arg Ala Tyr Ala Ala Ala Thr Gly Thr Ser Pro Leu

65 70 75 80

Pro Ser Gly Arg Asp Ser Tyr Arg Arg Leu Ala Asp Tyr Asn Asp Asp

85 90 95

Leu Gly Arg Met Ala Asp Gln Asn Pro Gly Leu Val Arg Lys Phe Thr

100 105 110

Leu Lys His Lys Ser Leu Glu Gly Lys Pro Val His Gly Val Glu Ile

115 120 125

Thr His Asp Val Thr Ala Val Asp Asp Gly Arg Pro Val Phe Leu Met

130 135 140

Met Gly Leu His His Ala Arg Glu Trp Pro Ser Gly Glu His Ala Ile

145 150 155 160

Glu Phe Ala His Asp Leu Val Arg Asn Tyr Gly Ser Asp Glu Arg Ile

165 170 175

Thr Ser Leu Leu Gln Lys Ala Arg Val Leu Val Val Pro Val Val Asn

180 185 190

Val Asp Gly Phe Glu Lys Ser Val Asn Asp Gly Gln Leu Ile Asp Leu

195 200 205

Arg Glu Ile Asp Asp Gly Gly Thr Gly Ser Ile Leu Ala Thr Pro Gly

210 215 220

Asn Ala Tyr Lys Arg Lys Asn Cys Arg Ile Val Asp Gly Leu Ser Pro

225 230 235 240

Val Ala Gly Glu Cys Ala Leu Ala Ser Ser Pro Gly Gly Phe Gly Ala

245 250 255

Gly Val Asp Leu Asn Arg Asn Tyr Gly Gly Phe Trp Gly Gly Pro Gly

260 265 270

Ala Ala Ala Glu Ser Val Gln Ala Thr Tyr Arg Gly Ala Ala Pro Phe

275 280 285

Ser Glu Pro Glu Thr Gln Asn Ile Arg Glu Leu Val Ser Ser Arg Gln

290 295 300

Val Thr Gly Leu Ile Thr Asn His Thr Phe Ser Asn Leu Val Leu Arg

305 310 315 320

Pro Asn Gly Val Ala Pro Asp Thr Val Gly Pro Asp Gly Gln Pro Ile

325 330 335

Gly Asn Pro Pro Asp Glu Ala Ala Leu Lys Glu Leu Gly Asp Arg Met

340 345 350

Ala Glu Gln Asn Gly Tyr Thr Ser Gln His Ser Trp Glu Leu Tyr Asp

355 360 365

Thr Thr Gly Thr Thr Glu Asp Trp Ser Tyr Asn Ala Thr Gly Gly Tyr

370 375 380

Gly Tyr Thr Phe Glu Ile Gly Pro His Glu Phe His Pro Pro Phe Pro

385 390 395 400

Glu Val Val Asp Glu Tyr Val Gly Ala Gly Glu Tyr Ala Gly Lys Gly

405 410 415

Asn Arg Glu Ala Phe Leu Leu Ala Leu Glu Ser Ala Val Asp Pro Glu

420 425 430

Ser His Ser Val Ile Ser Gly Lys Ala Pro Ala Gly Ala Thr Leu Arg

435 440 445

Leu Lys Lys Thr Phe Ala Thr Pro Thr Trp Ser Gly Thr Ile Lys Asp

450 455 460

Thr Leu Asp Thr Thr Met Thr Val Gly Ser Gly Gly Ser Tyr Thr Trp

465 470 475 480

His Val Asn Pro Ser Thr Arg Pro Val Val Lys Ala Arg Gln Ile Glu

485 490 495

Val Ile Gly Ser Glu Pro Leu Lys Arg Gln Thr Tyr Thr Gly Thr Thr

500 505 510

Ala Pro Gly Gln Pro Thr Glu Gln Glu Phe Val Val Asp Arg Asp Ala

515 520 525

Asp Val Phe Glu Ala Lys Leu Asp Trp Ala Thr Pro Asp Asp Leu Asp

530 535 540

Leu Tyr Val Leu Arg Lys Asn Ala Asp Gly Ser Leu Thr Gln Val Gly

545 550 555 560

Ser Ser Ala Gly Ser Val Gly Glu Lys Glu Arg Val Leu Leu Asp Asp

565 570 575

Pro Glu Gln Gly Thr Tyr Val Leu Arg Val Glu Asn Trp Ala Ser Val

580 585 590

Ala Pro Ser Trp Thr Leu Thr Ala Ser Leu Tyr Asp Ala Thr Val Asp

595 600 605

Glu Ile Gly Gly Val Ile Glu Asn Trp Thr Leu Ser Cys Glu Lys Asp

610 615 620

Gly Lys Val Leu Gln Gln Val Pro Val Val Val Asp Arg Gly Gln Arg

625 630 635 640

Val Lys Ala Asp Leu Lys Asn Cys Ala Lys Gly

645 650

Claims (11)

1.一种创新霉素(chuangxinmycin,CM)和/或开环创新霉素(secochuangxinmycin,SCM)的生物合成的基因簇，其特征在于，所述的基因簇依次包含cxnA、cxnB、cxnC、cxnD、cxnE、cxnF、cxnT、cxnR、trpRS九个关键功能基因，以及在创新霉素分子中引入S元素所必需的thiG、moeZ基因，其核苷酸序列分别为如SEQ ID No.2～12所示，包含cxnA、cxnB、cxnC、cxnD、cxnE、cxnF、cxnT、cxnR、trpRS九个关键功能基因的基因片段序列如SEQ ID No.1所示，

所述的生物合成基因簇中的各个基因所编码蛋白的具体功能为：

(1)CxnA为依赖于维生素B12的自由基S-腺苷甲硫氨酸家族的C-甲基转移酶，其功能为将吲哚丙酮酸的3-位甲基化，生成3-甲基吲哚丙酮酸；

(2)CxnB为氨基转移酶，其功能为将创新霉素的生物合成前体色氨酸(L-Trp)中的氨基转变为酮基，形成吲哚丙酮酸；

(3)CxnC为还原酶，其功能为将3-甲基吲哚丙酮酸的酮基还原为羟基，形成3-吲哚-2-羟基丁酸；

(4)CxnD为细胞色素P450酶，其功能为催化开环创新霉素(seco-chuangxinmycin,SCM)中的S原子和C原子形成C-S键，生成CM；

(5)CxnE为硫载体蛋白(sulfur carrier protein,SCP)，在JAMM金属蛋白酶家族的CxnF的作用下，C-末端10个氨基酸残基被水解露出双甘氨酸基序，而后先在SCP活化蛋白MoeZ(N-端为类似于SCP活化蛋白ThiF的结构域)作用下生成腺苷酰化的SCP，再在MoeZ的C-端硫氰酸酶结构域的作用下形成硫醇化的SCP，即为硫原子的供体；

(6)CxnF为JAMM金属蛋白酶家族成员，其功能为酶切CxnE的C-末端10个氨基酸残基，露出双甘氨酸基序；

(7)CxnR为转录调控蛋白，其功能为调节所述基因簇的表达；

(8)CxnT为创新霉素的转运蛋白；

(9)TrpRS为色氨酸tRNA合成酶，是创新霉素的自身抗性基因；

(10)ThiG为噻唑合成酶，其功能为将还原酶CxnC催化形成的3-吲哚-2-羟基丁酸中的羟基替换为由硫醇化SCP提供的巯基，得到3-吲哚-2-巯基丁酸(即CxnD的底物，开环创新霉素)；

(11)MoeZ为SCP活化蛋白，其作用为活化SCP蛋白CxnE，形成硫代羧酸酯，即硫醇化的SCP。

2.权利要求1所述的基因簇中的十一个关键基因编码的蛋白质。

3.一种开环创新霉素(SCM)，其结构如下式所示：

4.包含权利要求1所述的开环创新霉素或创新霉素基因簇的重组载体，优选的，所述的重组载体为包含酵母菌元件(ARSH4/CEN6复制子和TRP1筛选标记)、大肠杆菌元件(pUC ori复制子)和链霉菌元件(φC31整合酶及其整合位点attP、DNA接合转移起始位点oriT)的重组载体，最优选的，所述的重组载体为pCAP01。

5.转化了权利要求4所述的重组载体的宿主，优选的，所述的宿主为酵母、大肠杆菌或链霉菌，最优选的，所述的宿主为链霉菌。

6.通过微生物发酵生产创新霉素或权利要求3所述开环创新霉素(SCM)的方法，其步骤包括：

(1)将所述权利要求1所述的SCM的生物合成基因簇克隆至目标宿主，

(2)发酵权利要求5所述目标宿主并从发酵液中提取并纯化所述创新霉素或开环创新霉素SCM。

7.权利要求1所述的创新霉素或开环创新霉素生物合成基因簇和/或其编码蛋白在催化合成创新霉素、开环创新霉素或其类似物中的应用。

8.权利要求1所述的cxnR基因或其编码的CxnR蛋白在合成创新霉素、开环创新霉素中的应用。

9.一种提高创新霉素和/或开环创新霉素产量的方法，其特征在于，在创新霉素和/或开环创新霉素生产菌株中过表达权利要求1所述cxnR基因，所述的过表达的方法为，将高表达所述cxnR基因的重组载体转入所述生产菌株中，优选的，所述的重组载体为质粒pSET152，所述的生产菌株为来自中国药学微生物菌种保藏管理中心的菌株Actinoplanestsinanensis CPCC 200056(China Pharmaceutical Culture Collection)。

10.权利要求1所述的cxnD基因或其编码的CxnD蛋白在以开环创新霉素为底物合成创新霉素中的应用。

11.一种合成创新霉素的方法，其特征在于，以开环创新霉素为底物，发酵转化了所述cxnD基因的宿主，所述的宿主不包含所述创新霉素合成基因簇的其他基因，优选的，所述的宿主为链霉菌。