CN110484576B - 一种提高榴菌素和榴菌素b产量的方法 - Google Patents
一种提高榴菌素和榴菌素b产量的方法 Download PDFInfo
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Abstract
本发明公开了一种提高榴菌素和榴菌素B产量的方法。本发明是基于我们发现榴菌素和榴菌素B的生物合成途径会产生大量的早期支路产物，大量前体进入支路途径，而使得终产物榴菌素和榴菌素B的产量较低。本发明通过增加编码负责催化二环中间体形成的环化酶的基因拷贝数、更换强启动子等手段，提高榴菌素和榴菌素B生物合成中的二环中间体形成的效率，显著抑制支路途径，大幅减少支路产物，从而大幅提高榴菌素和榴菌素B的单位产量，同时使最终发酵产物的成分简化，榴菌素和榴菌素B的纯化更加容易，成本更低，能够促进榴菌素和榴菌素B的临床研究和应用。
Description
技术领域
本发明属于天然药物化学、微生物学、基因工程技术领域，具体涉及一种提高榴菌素和榴菌素B产量的方法。
背景技术
榴菌素（Granaticin）和榴菌素B是放线菌产生的次级代谢产物，是苯并异色满醌（benzoisochromanequinone，BIQ）类抗生素的重要成员，具有抗菌、抗肿瘤等多种生物活性，且作用机制新颖，是最具临床抗癌应用潜力的BIQ类化合物。榴菌素和榴菌素B能够特异地抑制法尼基转移酶（Farnesyltransferase，FTase）(The Journal of Antibiotics,2007, pp.1-12)、次黄嘌呤核苷酸脱氢酶（Inosine 5'-monophosphate dehydrogenase，IMPDH）(中国药学会学术年会暨中国药师周, 2008, pp.539-44)以及细胞分裂周期蛋白激酶7（cell division cycle 7 kinase, Cdc7）(WO2011112635A1)，而这些酶是目前发展抗肿瘤新药的重要靶标(Mini Reviews in Medicinal Chemistry, 2009, pp.638-52;Current Opinion in Drug Discovery and Development, 2006, pp.595-605; EuropeanJournal of Cancer, 2010, pp.33-40)。目前，全球范围内已有多个利用榴菌素或榴菌素B治疗癌症及其它疾病的专利（CN102844023A，WO2011112635A1，CN107349199A）。
榴菌素最早在橄榄色链霉菌（Streptomyces olivaceus）中被分离发现(Helvetica Chimica Acta, 1957, pp.1262-69)。此后，发现紫红链霉菌（S. violaceoruber）(Helvetica Chimica Acta, 1966, pp.1736-40)、嗜热的热紫链霉菌渗透亚种（S. thermoviolaceus subsp. pingens）WR-141(Archivum immunologiae ettherapiae experimentalis, 1969, pp.827-32)、玫瑰球孢链霉菌榴菌素变种（S. globispororoseus var. granaticus）(Antibiotiki, 1976, pp.582-6)、砖红链霉菌（S. lateritius）(Journal of Basic Microbiology, 1980, pp.543-51)以及一个无效命名种“产石蕊链霉菌”（“S. litmogenes”）(The Journal of Antibiotics, 1975, pp.156-56)等菌株均能产生榴菌素，我们报道的链霉菌新种，粤蓝链霉菌（S. vietnamensis）GIMV4.0001(International Journal of Systematic and Evolutionary Microbiology,2007, pp.1770-74)，也被发现能产生榴菌素(Antonie Van Leeuwenhoek, 2011, pp.607-17)。产榴菌素的菌株通常也能产生榴菌素B，二者在结构上的差别在于榴菌素B在榴菌素的糖基上通过C-O糖苷键连接了额外一个玫红糖，二者来源于同一生物合成途径。
大量、低成本的获得目标活性物质是进行前期研究、临床试验和应用的前提。榴菌素含有两个手性中心以及糖基与母核之间通过两个碳碳单键进行环化的特殊结构，使得化学合成较为复杂，目前虽已有以四氢萘酮（tetralone）为反应起始物进行榴菌素全合成的报道(Journal of the American Chemical Society, 1987, pp.3402-08)，但整个合成需要20个步骤，得率低，且产物是多种光学异构的混合物，成本高无法量产，而榴菌素B则目前还没有全合成的报道。因此，微生物发酵生产榴菌素和榴菌素B是当前可行的方法。
发明内容
本发明的目的在于提供一种提高榴菌素和榴菌素B产量的方法。
本发明采取的技术方案如下：
一种提高榴菌素和榴菌素B产量的方法，其特征在于，是通过提高榴菌素和榴菌素B生物合成中的二环中间体形成的效率实现的；
所述的二环中间体的结构式如式1所示：
式1。
优选，所述的提高榴菌素和榴菌素B生物合成中的二环中间体形成的效率，是通过以下任一方法或任意方法的组合实现：
A、在产生菌的基因组中增加编码负责催化二环中间体形成的环化酶的基因拷贝数；
B、将产生菌的基因组中催化二环中间体形成的环化酶的基因自身天然启动子，更换成更强的启动子；
C、将催化二环中间体形成的环化酶进行定向改造，以使该环化酶的催化效率提高，并将能翻译出改造后的环化酶的基因导入产生菌的基因组中；
所述的产生菌是产生榴菌素和榴菌素B的菌株。
优选，所述的催化二环中间体形成的环化酶的氨基酸序列如SEQ ID NO.1所示。
优选，所述的环化酶的编码基因的核苷酸序列如SEQ ID NO.2所示。
一种产榴菌素和榴菌素B的工程菌，其特征在于，是对产榴菌素和榴菌素B的产生菌进行以下任一方法或任意的组合的改造：
A、在产生菌的基因组中增加编码负责催化二环中间体形成的环化酶的基因拷贝数；
B、将产生菌的基因组中催化二环中间体形成的环化酶的基因自身天然启动子，更换成更强的启动子；
C、将催化二环中间体形成的环化酶进行定向改造，以使该环化酶的催化效率提高，并将能翻译出改造后的环化酶的基因导入产生菌的基因组中；
所述的二环中间体的结构式如式1所示：
式1。
优选，所述的产生菌为紫红链霉菌S. violaceoruber DMS 40701、砖红链霉菌 S. lateritius NBRC 12788或粤蓝链霉菌S. vietnamensis GIMV4.0001。更优选，所述的产生菌为粤蓝链霉菌S. vietnamensis GIMV4.0001。
本发明的有益效果为：
（1）本发明通过提高榴菌素和榴菌素B生物合成中的二环中间体合成的效率，使得产生菌中原本存在的早期支路产物途径得到显著抑制，早期支路产物大幅减少，而作为终产物的榴菌素和榴菌素B的产量大幅提高。
（2）本发明通过抑制支路产物的产生，而大幅提高了目标终产物榴菌素和榴菌素B的产量，使得最终发酵产物的成分简化，使榴菌素和榴菌素B的纯化更加容易，成本更低。
本发明的粤蓝链霉菌(Streptomyces vietnamensis) GIMV4.0001，现在在国家知识产权局指定的保藏单位保藏，保藏日期为2005年12月9日，保藏单位名称：中国典型培养物保藏中心，保藏号CCTCC NO：M 205143，该菌株公开于专利CN 200610034149.7中。
附图说明
图1 为三株榴菌素和榴菌素B产生菌的产量LC-MS分析比较。其中，DMS 40701为紫红链霉菌（S. violaceoruber）DMS 40701，NBRC 12788为砖红链霉菌（S. lateritius）NBRC12788，GIMV4.0001为粤蓝链霉菌（S. vietnamensis） GIMV4.0001。
图2 为简化的榴菌素和榴菌素B生物合成途径及其早期支路产物。其中，百分比显示的是中间体进入支路或终产物途径的比例。KR，酮基还原酶；ARO，芳香化酶；bicyclicintermediate，二环中间体；1，榴菌素；2，榴菌素B；3，SEK34；4，SEK34b；5，mutactin；6，二氢榴菌素B；7，dehydromutactin，8，EM18；9，GTRI-02。
图3为粤蓝链霉菌GIMV4.0001代谢谱动态变化LC-MS分析图。其中，1，榴菌素；2，榴菌素B；3，SEK34；4，SEK34b；5，mutactin；6，二氢榴菌素B；7，dehydromutactin，8，EM18；9，GTRI-02。
图4 为粤蓝链霉菌经改造后的K33-1与野生株GIMV4.0001产榴菌素和榴菌素B的液相分析比较。其中，菌株K33-1是在野生株的基因组中导入一个带有强启动子的环化酶基因gra-orf33。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1 三株榴菌素和榴菌素B产生菌的产量分析比较
以YEME液体培养基（酵母提取物3 g、蛋白胨5 g、麦芽提取物3 g、葡萄糖10 g，补水至1 L，pH 7.2，121℃灭菌20 min后加入2.5 mol/L MgCl2 2 mL）为种子培养基。将紫红链霉菌（S. violaceoruber）DMS 40701、砖红链霉菌（S. lateritius）NBRC 12788、粤蓝链霉菌（S. vietnamensis） GIMV4.0001孢子分别接种于YEME液体培养基中，于28℃，180 rpm震荡培养2天作为种子液。将种子液按5% v/v 接种量接种于高氏合成一号液体培养基中（可溶性淀粉20 g、KNO3 1 g 、K2HPO4 0.5 g、MgSO4·7H2O 0.5 g、NaCl 0.5 g、FeSO4 0.01g，补水至1L，pH 7.2，121℃灭菌20 min），于28℃，180 rpm震荡培养7天。发酵结束后，发酵液用等体积乙酸乙酯萃取3次，有机相减压蒸干，用甲醇重新溶解，进行液质(LC-MS)分析。在相同发酵条件下，粤蓝链霉菌（S. vietnamensis）GIMV4000.1产榴菌素和榴菌素B的能力最强，砖红链霉菌（S. lateritius）NBRC 12788次之，而紫红链霉菌（S. violaceoruber）DMS 40701产量最低（图1）。
实施例2 榴菌素和榴菌素B产生菌粤蓝链霉菌的次级代谢产物分析
以YEME液体培养基（酵母提取物3 g、蛋白胨5 g、麦芽提取物3 g、葡萄糖10 g，补水至1 L，pH 7.2，121℃灭菌20 min后加入2.5 mol/L MgCl2 2 mL）为种子培养基。将粤蓝链霉菌（S. vietnamensis）GIMV4.0001孢子接种于YEME液体培养基中，于28℃，180 rpm震荡培养2天作为种子液。将种子液按5% v/v接种量接种于高氏合成一号液体培养基中（可溶性淀粉20 g、KNO3 1 g 、K2HPO4 0.5 g、MgSO4·7H2O 0.5 g、NaCl 0.5 g、FeSO4 0.01 g，补水至1 L，pH 7.2，121℃灭菌20 min），于28℃，180 rpm震荡培养7天，共发酵20 L。
发酵结束后，发酵液用等体积乙酸乙酯萃取3次，有机相减压旋蒸，获得粗提物12g。粗提物过常压硅胶柱，流动相用二氯甲烷/甲醇体系，二者比例设置为100:0，98:2，96:4，94:6，92:8，90:10，80:20，70:30，50:50，0:100 v/v十个梯度，每个梯度用3个柱体积进行洗脱。洗脱所得馏分进行TLC分析，根据成分相似度进行合并，获得11个馏分段。对各馏分段利用制备液相、半制备液相、琼脂糖凝胶柱等进行分离纯化，最终，除榴菌素（1）、榴菌素B（2）、二氢榴菌素B（6）外，还获得6个来源于榴菌素生物合成途径的早期支路产物，分别是SEK34(3)，SEK34b (4)，mutactin (5)，dehydromutactin (7)，EM18 (8)和GTRI-02 (9)（图2）。化合物3，4，5，7，8，9的核磁数据如下：
化合物 3, SEK34: 1H NMR (700 MHz, DMSO-d 6): δ H = 7.51 (t, J = 7.7 Hz,1H), 6.99 (br s, 1H), 6.97 (d, J = 7.7 Hz, 1H), 6.91 (d, J = 7.7 Hz, 1H),5.54 (s, 1H), 5.18 (d, J = 2.1 Hz, 1H), 4.22 (d, J = 16.1 Hz, 1H), 4.13 (d, J= 16.1 Hz, 1H), 3.06 (d, J = 16.1 Hz, 1H), 2.65 (d, J = 15.4 Hz, 1H), 1.60(s, 3H) ppm; 13C NMR (175 MHz, DMSO-d 6): δ C = 193.1 (C), 170.6 (C), 165.6 (C),163.9 (C), 159.8 (C), 136.2 (CH), 135.0 (CH), 124.6 (CH), 118.5 (C), 118.0(CH), 100.9 (C), 99.7 (CH), 88.2 (CH), 49.6 (CH2), 37.5 (CH2), 27.5 (CH3) ppm。
化合物 4, SEK34b: 1H NMR (700 MHz, DMSO-d 6): δ H = 7.73 (dd, J = 8.4,7.0 Hz, 1H), 7.57 (dd, J = 8.4, 1.4 Hz, 1H), 7.31 (d, J = 7.0 Hz, 1H), 6.17(s, 1H), 5.48 (d, J = 2.1 Hz, 1H), 5.16 (d, J = 2.1 Hz, 1H), 4.44 (s, 2H),2.36 (s, 3H) ppm; 13C NMR (175 MHz, DMSO-d 6): δ C = 178.4 (C), 170.5 (C), 165.8(C), 165.5 (C), 163.8 (C), 157.4 (C), 135.7 (C), 133.4 (CH), 128.6 (CH),120.9 (C), 118.1 (C), 111.1 (CH), 99.5 (CH), 88.1 (CH), 37.3 (CH2), 19.5 (CH3)ppm。
化合物 5, mutactin: 1H NMR (600 MHz, DMSO-d 6) :δ H = 12.66 (s, 1H),6.81 (s, 1H), 6.18 (d, J = 2.4 Hz, 1H), 5.41 (d, J = 2.4 Hz, 1H), 4.23 (m,1H), 2.99 (dd, J = 16.8, 3.6 Hz, 1H), 2.92 (dd, J = 16.8, 3.6 Hz, 1H), 2.76(dd, J = 16.8, 6.6 Hz, 1H), 2.67 (dd, J = 16.8, 6.6 Hz, 1H), 2.21 (s, 3H)ppm; 13C NMR (150 MHz, DMSO-d 6) :δC =204.4 (C), 170.1 (C), 163.9 (C), 162.3(C), 159.7 (C), 146.6 (C), 141.9 (C), 124.1(C), 114.6 (C), 116.3(CH), 104.8(CH), 89.6 (CH), 64.4 (CH), 46.2 (CH2), 35.7 (CH2), 20.3 (CH3) ppm。
化合物 7, dehydromutactin: 1H NMR (600 MHz, DMSO-d 6) :δ H = 11.54 (brs, 2H), 7.29 (m, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 7.8 Hz, 1H),6.68 (s, 1H), 6.18 (d, J = 1.8 Hz, 1H), 5.43 (d, J = 2.4 Hz, 1H), 2.27 (s,3H) ppm. 13C NMR (150 MHz, DMSO-d 6) :δ C = 170.2 (C), 164.3 (C), 161.1 (C),155.9 (C), 154.6 (C), 136.6 (C), 134.9 (C), 128.2 (CH), 119.9 (C), 115.3(CH), 112.9 (C), 110.4 (CH), 108.2 (CH), 105.0 (CH), 89.4 (CH), 19.8 (CH3)ppm。
化合物 8, EM18: 1H NMR (700 MHz, DMSO-d 6): δ H = 12.34 (s, 1H), 7.51(t, J = 7.7 Hz, 1H), 6.88 (d, J = 7.7 Hz, 1H), 6.84 (d, J = 7.7 Hz, 1H), 6.21(d, J = 2.1 Hz, 1H), 5.23 (d, J = 2.1 Hz, 1H), 4.10 (s, 1H), 3.08 (d, J =17.5 Hz, 1H), 2.67 (d, J = 17.5 Hz, 1H), 1.25 (s, 3H) ppm; 13C NMR (175 MHz,DMSO-d 6): δ C = 204.3 (C), 170.5 (C), 163.8 (C),163.2 (C), 161.4 (C), 141.8(C), 136.9 (CH), 120.6 (CH), 116.3 (CH), 116.0 (C), 102.7 (CH), 89.0 (CH),70.9 (C), 54.5 (CH), 48.5 (CH2), 27.4 (CH3) ppm。
化合物 9, GTRI-02: 1H NMR (600 MHz, DMSO-d 6) : δ H = 6.60 (s, 1H), 4.13(m, 1H), 3.06 (dd, J = 16.2, 3.6 Hz, 1H), 2.81 (dd, J = 16.2, 7.2 Hz, 1H),2.74 (dd, J = 15.6, 3.0 Hz, 1H), 2.49 (dd, J = 16.2, 7.2 Hz, 1H), 2.39 (s,3H), 2.32 (s, 3H) ppm; 13C NMR (150 MHz, DMSO-d 6) : δ C = 205.3 (C), 197.0 (C),157.0 (C), 145.3 (C), 137.5 (C), 130.8 (C), 123.2 (C), 113.6 (CH), 64.7 (CH),49.2 (CH2), 39.3 (CH2), 32.3 (CH3), 18.4 (CH3) ppm。
为进一步了解粤蓝链霉菌在整个发酵周期中代谢谱变化特征，特别是榴菌素和榴菌素B以及相关支路产物的变化特征，对发酵周期内每天的次级代谢产物谱利用LC-MS进行跟踪分析，发现在发酵第一天即可检测到榴菌素、榴菌素B以及SEK34（3）的产生，而在发酵第二天即可检测到其它所有上述化合物的存在。发酵早期有大量SEK34（3）的产生，在发酵后期SEK34b（4）的含量也迅速增加，而其它支路产物在整个发酵周期中含量相对较低（图3）。
为了进一步了解榴菌、榴菌素B与几个主要支路产物产量水平及比例关系，利用纯的各化合物进行曲线标定，计算出榴菌素、榴菌素B、SEK34、SEK34b的产量分别为：34 mg/L、193 mg/L、105 mg/L、120 mg/L。以摩尔浓度计，一环中间体有约65%进入了SEK34/SEK34b支路产物途径，而仅有约35%进入了榴菌素和榴菌素B途径。
实施例3 将带有强启动子的编码催化形成二环中间体的环化酶基因gra-orf33导入粤蓝链霉菌基因组中
根据已有文献的报道(Chemistry & Biology, 1998, pp.647-59)，gra-orf33可能编码了催化形成二环中间体的环化酶（环化酶的氨基酸序列如SEQ ID NO.1所示，其编码基因的核苷酸序列如SEQ ID NO.2所示）。利用引物orf33F(5′- ACTAGT TCGAGGAGGAGACCCACATGACC-3′，下划线为酶切位点)/orf33R(5′- GAATTC GAACCCGCCGGGCGCTCA-3′，下划线为酶切位点)从粤蓝链霉菌（S. vietnamensis）GIMV4.0001基因组中扩增出榴菌素生物合成基因簇中gra-orf33基因953 bp的片段，克隆至载体pSET-KasO*(pBS21003)( 该质粒是衍自质粒pSET152，是插入了KasO*这个强启动子的载体，具体构建方法参见PNAS, 2017, 114 (52) E11131-E1114)，获得重组质粒pSET-K-33。将质粒pSET-K-33通过属间接合转移转入粤蓝链霉菌，在含萘啶酮酸和安普霉素（Apr）抗性的YD平板上筛选接合子。随机接合子扩繁，提取基因组DNA，利用引物ApraF（5′-GGTCCACAGCTCCTTCCGTA-3′）/ApraR（5′-TTATGAGCTCAGCCAATCGAC-3′）进行PCR验证。电泳检测显示，接合子能扩增出708 bp的安普霉素抗性基因片段，而对照野生株无任何扩增条带，与预期相符，提示gra-orf33基因成功导入粤蓝链霉菌，将新菌株命名为粤蓝链霉菌K33-1。
实施例4 利用菌株K33-1发酵生产榴菌素和榴菌素B
以YEME液体培养基（酵母提取物3 g、蛋白胨5 g、麦芽提取物3 g、葡萄糖10 g，补水至1 L，pH 7.2，121℃灭菌20 min后加入2.5 mol/L MgCl2 2 mL）为种子培养基。将粤蓝链霉菌K33-1孢子接种于YEME液体培养基中，于28℃，180 rpm震荡培养2天作为种子液。将种子液按5% v/v接种量接种于高氏合成一号液体培养基中（可溶性淀粉20 g、KNO3 1 g 、K2HPO4 0.5 g、MgSO4·7H2O 0.5 g、NaCl 0.5 g、FeSO4 0.01 g，补水至1 L，pH 7.2，121℃灭菌20 min），于28℃，180 rpm震荡培养7天。以粤蓝链霉菌野生株（S. vietnamensis）GIMV4000.1作为对照，同批发酵。发酵结束后，发酵液用等体积乙酸乙酯萃取3次，有机相减压蒸干，用甲醇重新溶解，进行液相分析。在相同发酵条件下，与粤蓝链霉菌野生株GIMV4000.1相比，粤蓝链霉菌K33-1榴菌素和榴菌素B的产量显著增加，且主要支路产物SEK34和SEK34b几乎检测不到（图4），说明额外增加一个带有强启动子的gra-orf33基因拷贝，能够显著抑制SEK34/SEK34b支路途径，而强化榴菌素和榴菌素B的终产物途径。经定量分析，粤蓝链霉菌K33-1菌株榴菌素和榴菌素B的产量分别达到120 mg/L和495 mg/L，而同批发酵的粤蓝链霉菌野生株GIMV4000.1榴菌素和榴菌素B的产量分别为98 mg/L和115 mg/L。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东省微生物研究所（广东省微生物分析检测中心）
<120> 一种提高榴菌素和榴菌素B产量的方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 306
<212> PRT
<213> 粤蓝链霉菌GIMV4.0001(Streptomyces vietnamensis GIMV4.0001)
<400> 1
Met Thr Gly Ile Thr Ala Gly Ala Val Arg Thr Glu Leu Thr Glu Val
1 5 10 15
Ala Asp Gly Val Phe Ala His Val Gln Pro Asp Gly Gly Trp Cys Leu
20 25 30
Asn Asn Ala Gly Leu Val Val Ser Gly Asp Arg Ala Ala Leu Ala Asp
35 40 45
Thr Ala Ala Thr Glu Ala Arg Ala Arg Thr Leu Arg Glu Val Val Leu
50 55 60
Arg Val Ala Pro Ala Ala Pro Gln Val Leu Val Asn Thr His Phe His
65 70 75 80
Gly Asp His Thr Phe Gly Asn Phe Val Phe Pro Glu Ser Leu Val Val
85 90 95
Gly His Glu Arg Thr Arg Thr Glu Met Thr Glu Thr Gly Leu His Met
100 105 110
Thr Gly Leu Trp Pro Asp Val Glu Trp Gly Asp Leu Arg Leu Val Pro
115 120 125
Pro Ala Leu Thr Phe Arg Asp Arg Leu Thr Leu His Ile Gly Glu Lys
130 135 140
Thr Ala Glu Leu Leu His Leu Gly Pro Ala His Thr Ser Asn Asp Thr
145 150 155 160
Val Leu Trp Leu Pro Ala Glu Arg Val Leu Phe Thr Gly Asp Leu Val
165 170 175
Met Asn Gly Val Thr Pro Phe Cys Pro Met Gly Ser Val Ala Gly Ser
180 185 190
Leu Glu Ala Leu Asp Thr Met Arg Ala Leu Ala Pro Glu Val Val Val
195 200 205
Pro Gly His Gly Pro Val Ala Gly Pro Gly Val Phe Asp Glu Thr Glu
210 215 220
Gly Tyr Leu Arg Leu Leu Gln Asp Leu Ala Glu Gln Gly Leu Ala Glu
225 230 235 240
Ser Leu Asp Pro Val Glu Leu Ala Arg Arg Thr Asp Leu Gly Glu Tyr
245 250 255
Ala Arg Trp Leu Asp Ala Glu Arg Leu Val Pro Asn Leu Phe Arg Ala
260 265 270
Tyr Ala Glu Lys Gln Gly Glu Pro Arg Gly Ser His Val Asp Met Ser
275 280 285
Glu Leu Phe Ala Arg Met Ile Asp Tyr His Gly Gly Leu Pro Thr Cys
290 295 300
His Ala
305
<210> 2
<211> 921
<212> DNA
<213> 粤蓝链霉菌GIMV4.0001(Streptomyces vietnamensis GIMV4.0001)
<400> 2
atgaccggaa tcaccgccgg cgccgtccgg accgagctga cggaggtcgc cgacggggtg 60
ttcgcccacg tccagccgga cgggggctgg tgcctgaaca acgccggtct cgtcgtctcg 120
ggcgaccgcg ccgcgctcgc ggacaccgcg gccaccgagg cgcgcgccag gaccctgcgc 180
gaggtggtgc tccgggtcgc cccggccgcc ccacaggtcc tggtcaacac ccacttccac 240
ggcgaccaca ccttcggcaa cttcgtcttc ccggagtccc tcgtcgtcgg ccacgagcgc 300
acccgtacgg agatgaccga gacgggcctg cacatgaccg gcctgtggcc ggacgtggag 360
tggggcgacc tgcggctcgt cccgccggcg ctgaccttcc gcgaccggct caccctgcac 420
atcggcgaga agacggccga gctgctgcac ctgggcccgg cgcacaccag caacgacacg 480
gtgctctggc tgccggccga acgggtgctg ttcaccggcg acctggtgat gaacggggtg 540
acgccgttct gccccatggg ctcggtcgcc ggctccctcg aagccctgga cacgatgcgc 600
gccctcgccc cggaggtggt cgtccccggc cacggcccgg tcgcgggccc cggcgtgttc 660
gacgagaccg agggctatct gcgcctgctc caggacctcg ccgagcaggg gctcgccgag 720
tccctcgacc cggtcgaact ggcccgccgc accgacctgg gcgagtacgc gcgctggctc 780
gacgccgagc ggctcgtccc caacctcttc cgcgcgtacg ccgagaaaca gggcgagccc 840
cgcggttccc acgtggacat gagcgaactc ttcgcccgca tgatcgacta ccacggcggc 900
ctgcccacct gccacgcctg a 921
Claims (3)
1.一种提高榴菌素和榴菌素B产量的方法，其特征在于，是通过提高榴菌素和榴菌素B生物合成中的二环中间体形成的效率实现的；
所述的二环中间体的结构式如式1所示：
式1
所述的提高榴菌素和榴菌素B生物合成中的二环中间体形成的效率，是通过将产生菌的基因组中催化二环中间体形成的环化酶的基因拷贝数增加或/和将该基因自身天然启动子更换成更强的启动子实现，所述的催化二环中间体形成的环化酶的氨基酸序列如SEQ IDNO.1所示，所述的产生菌为粤蓝链霉菌S. vietnamensis GIMV4.0001。
2.根据权利要求1所述的提高榴菌素和榴菌素B产量的方法，其特征在于，所述的环化酶的编码基因的核苷酸序列如SEQ ID NO.2所示。
3.一种产榴菌素和榴菌素B的工程菌，其特征在于，是对产榴菌素和榴菌素B的产生菌进行以下改造：将产生菌的基因组中催化二环中间体形成的环化酶的基因拷贝数增加或/和将该基因自身天然启动子更换成更强的启动子实现，所述的催化二环中间体形成的环化酶的氨基酸序列如SEQ ID NO.1所示，所述的产生菌为粤蓝链霉菌S. vietnamensisGIMV4.0001；
所述的二环中间体的结构式如式1所示：
式1。
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