CN113801802B - 一种链霉菌及其应用 - Google Patents
一种链霉菌及其应用 Download PDFInfo
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- CN113801802B CN113801802B CN202010542053.1A CN202010542053A CN113801802B CN 113801802 B CN113801802 B CN 113801802B CN 202010542053 A CN202010542053 A CN 202010542053A CN 113801802 B CN113801802 B CN 113801802B
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- Prior art keywords
- rubicin
- beta
- fermentation
- resin
- streptomyces
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Abstract
本发明公开了一株链霉菌，所述链霉菌为Streptomyces.sp.CB00271，保藏编号为CCTCC M 2020176。本发明通过特异性基因探针进行基因组挖掘获得了β‑玉红霉素的高产菌株，并且其玉红霉素基因簇和报道的不一样，有其独特的新颖性，预测可以产生新的玉红霉素衍生物。Streptomyces.sp.CB00271除了产生β‑玉红霉素外，还产生多种玉红霉素类似物，如γ‑玉红霉素，3‑羟基‑β‑玉红霉素，DK‑7814‑B(IV)和数个未知类似物，其β‑玉红霉素的发酵产率达到130 mg/L左右，相比于已报道的最高产量43 mg/L，提高了近3倍。
Description
技术领域
本发明涉及一种链霉菌及其应用，特别涉及一种制备玉红霉素及其衍生物的高产链霉菌，尤其是涉及一种制备β-玉红霉素及其衍生物的高产链霉菌，属于生物医药技术领域。
背景技术
玉红霉素(rubromycins，Rubs)类天然产物具有一个5,6螺环缩酮的核心骨架。已发现的20个类似物中，萘醌结构可分为两种类型：一种是萘醌的醌环和5,6螺环缩酮相连，如β-玉红霉素；另一种是萘醌的苯环和5,6螺环缩酮相连，如γ-玉红霉素。玉红霉素类天然产物中绝大部分都是γ-玉红霉素型的骨架，目前只有3个β-玉红霉素型的结构。
玉红霉素类天然产物具有广泛的生物活性，如抗菌活性，细胞毒性和逆转录酶抑制活性等，尤其是部分化合物表现出良好的对人类端粒酶的抑制活性。通过玉红霉素类天然产物构效关系的研究，5,6螺环缩酮结构正是玉红霉素类天然产物的主要药效团，萘醌结构和末端基团对其活性也有一定影响，其中具有独特萘醌结构的β-玉红霉素具有最好的人类端粒酶抑制活性，可能跟其萘醌结构和末端甲酯化有关。β-玉红霉素可由不同的放线菌产生，但其产量普遍较低，未超过50mg/L，而且在大多数的溶剂中溶解度极低，极大限制了β-玉红霉素作为以端粒酶为靶点的新一代抗肿瘤药物的开发。
随着基因组测序的爆发式增长和基因组挖掘技术的成熟，基因组挖掘技术在发现新天然产物和发现有药用开发价值天然产物的高产菌株等研究中起到了关键性的推动作用。玉红霉素类天然产物含有的独特5,6螺环缩酮结构也正是其药效基团。有关报道表明，grhO6与玉红霉素类化合物灰紫红素(griseorhodin)中的5,6螺环缩酮的形成密切相关，而与灰紫红素结构及其类似的玉红霉素其生物合成基因簇中也存在和grhO6高度同源的基因rubN。因此，玉红霉素类化合物的生物合成基因簇中应含有一个和rubN高度同源的保守序列以负责其核心5,6螺环缩酮结构的合成，为我们进行基因组挖掘提供了很好的探针。
β-玉红霉素是通过活性筛选从山丘链霉菌(Streptomyces collinus)的发酵产物中分离发现的一种新型5,6螺环缩酮类抗生素，其结构如下所示：
β-玉红霉素因其复杂独特的分子结构，无法通过传统的化学合成方法来获取，通过微生物发酵是目前制备β-玉红霉素最切实可行的手段。此外，β-玉红霉素从发酵液中的分离纯化仍采用乙酸乙酯萃取发酵上清液等传统手段，步骤繁多且过程中需要消耗大量的有机溶剂。因此，寻找β-玉红霉素高产菌株，并对其进行系统地发酵培养优化和建立规模化的发酵调控工艺，可以大幅提高β-玉红霉素产量，在此基础上通过高效的分离纯化方法可以获得足量的β-玉红霉素，以有效地解决其现阶段的发展瓶颈，加速推动β-玉红霉素后继的抗癌活性分析和作用机理等临床前期研究，以及抗肿瘤新药的应用开发。
发明内容
本发明解决的技术问题是，提供一种玉红霉素的产生菌，进一步提高玉红霉素的产量，以满足工业化生产的应用需求。
本发明通过对比和分析β-玉红霉素及其它玉红霉素类天然产物的生物合成基因簇，确定合适的基因组挖掘的探针RubN，对微生物菌种数据库进行基因组挖掘，最终筛选获得高产β-玉红霉素的新产生菌株CB00271，并建立和优化了β-玉红霉素的微生物发酵制备工艺。
为了解决上述技术问题，本发明的技术方案如下：
一株链霉菌，所述链霉菌为Streptomyces sp.CB00271，已于2020年6月2日保藏在中国典型培养物保藏中心，保藏编号为CCTCC M 2020176。
本发明还提供了上述链霉菌在制备玉红霉素及其衍生物的应用，所述玉红霉素及其衍生物的结构式为：
其中，R1和R2选自H、OH；
或
具体，本发明所述玉红霉素及其衍生物包括β-玉红霉素，γ-玉红霉素，3-羟基-β-玉红霉素以及DK-7814-B。
优选的，所述玉红霉素及其衍生物为β-玉红霉素，所述β-玉红霉素的结构式为：
优选的，所述链霉菌制备β-玉红霉素及其衍生物的过程，包括以下步骤：
S1、将CB00271发酵7天后，离心收集发酵后的树脂和菌体，用二氯甲烷和甲醇混合溶剂对树脂和菌体进行多次超声提取，直至树脂提取液无明显颜色，合并提取液，得到混合液；
S2、将混合液进行浓缩干燥，进行硅胶色谱柱分离，最后用二氯甲烷和甲醇混合溶剂重新溶解分离产物，得到β-玉红霉素及其衍生物。
优选的，二氯甲烷和甲醇混合溶剂为二氯甲烷和甲醇按体积比1:1混合。
优选的，HPLC分析条件及程序为：流动相A为99.9％去离子水和0.1％甲酸；流动相B为100％乙腈，流速为1.0mL/min，紫外检测器波长为310nm，线性梯度分析程序为：0-1分钟，95％A；1-18分钟，95％A至5％A；18-20分钟，5％A；20-20.5分钟，5％A至95％A；20.5-25分钟，95％A。
优选地，发酵工艺使用的培养基中，以可溶性淀粉和葡萄糖作为碳源；以蛋白胨和酵母粉为氮源。
优选地，在发酵过程中，每升发酵液接种量为2％-10％。
优选地，在发酵过程中，每升发酵液接种量为3％-6％。
优选地，在发酵过程中，每升发酵液接种量为4％。
所述每升发酵液接种量过多过少均会影响产物的产量。接种量过多会导致菌体初期密度太大，代谢产物负反馈效应导致菌体提前死亡，从而导致产量降低；接种量过少会导致菌体初期密度低，产能降低。
优选地，在发酵过程中添加大孔吸附树脂。
优选地，所述的大孔吸附树脂包括HP20，DA-201，XAD-8，D-201，XAD-8。
优选地，所述大孔吸附树脂为HP20。
相比于其他大孔树脂，HP20易处理，可回收重复利用。
优选地，每升发酵液中添加树脂为培养基总重量的1％-10％。
优选地，每升发酵液中添加树脂为培养基总重量的1％-3％。
优选地，每升发酵液中添加树脂为培养基总重量的1％。
所述每升树脂添加量过多过少均会影响产物的产量。树脂添加过多，会影响菌体的生长，导致菌体密度降低，影响产量；树脂添加过少，当树脂吸附饱和后，无法继续吸附目标产物，负反馈调节将影响菌体的生长，进而导致产量降低。
优选地，发酵周期为5-7天。
优选地，发酵工艺使用的培养基的pH值为6.5-7.5。
优选地，按重量份计，所述发酵的培养基的组成包括：5份可溶性淀粉，5份葡萄糖，2.5份蛋白胨，2.5份酵母粉，0.5份酸水解酪蛋白，0.3份丙酮酸钠，0.25份MgSO4·7H2O，0.5份K2HPO4，pH 5.0-7.0，并在每升培养基中添加大孔吸附树脂。
本发明中，筛选上述菌株的具体步骤如下：
S1、玉红霉素类天然产物生物合成基因簇的比较分析，确定基因组挖掘探针；
S2、通过基因探针对微生物数据进行基因组挖掘，筛选到可获得的玉红霉素类产生菌；
S3、对筛选到的玉红霉素类产生菌进行进一步地基因簇和发酵分析，确定链霉菌CB00271为新的β-玉红霉素产生菌；
S4、对链霉菌CB00271进行培养基和发酵条件的优化，得到β-玉红霉素的产量最优的条件。
与现有技术相比，本发明的有益效果如下：
1.本发明通过基因组挖掘获得了β-玉红霉素的新产生菌CB00271，并且其玉红霉素基因簇和报道的不一样，有其独特的新颖性，预测可以产生新的玉红霉素衍生物。
2.本发明所获得的β-玉红霉素产生菌CB00271，除了产生β-玉红霉素外，还产生多种玉红霉素类似物，如γ-玉红霉素，3-羟基-β-玉红霉素，DK-7814-B和数个未知类似物。
3.本发明通过基因组挖掘和后期培养基及发酵条件的优化获得了β-玉红霉素的高产菌株CB00271，其β-玉红霉素的发酵产率达到130mg/L左右。
附图说明
图1表示发酵产物β-玉红霉素的化学结构式；
图2表示β-玉红霉素高产菌株(CB00271)的筛选流程图；
图3表示CB00271玉红霉素基因簇和报道的玉红霉素基因簇对比分析；
图4表示β-玉红霉素产生菌株(CB00271)种属分析；
图5表示β-玉红霉素的分析数据图；
图6表示γ-玉红霉素的分析数据图；
图7表示3-羟基-β-玉红霉素的分析数据图；
图8表示DK-7814-B的分析数据图；
图9表示CB00271在不同培养基中β-玉红霉素的发酵产量；
图10表示CB00271在mR2A培养基中不同接种量β-玉红霉素的发酵产量；
图11表示CB00271在mR2A培养基中不同HP20添加量β-玉红霉素的发酵产量。
具体实施方式
以下将结合实施例来详细说明本发明。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
实施例1：玉红霉素类天然产物生物合成基因簇分析及探针确定
通过对比分析已报道的玉红霉素类生物合成基因簇：β-玉红霉素生物合成基因簇rub(序列号.AF293355)、灰紫红菌素生物合成基因簇grh(序列号.AF509565)和hyaluromycin生物合成基因簇hya(序列号.AF509565)，发现这三种基因簇的相似度很高，说明玉红霉素类化合物的生物合成具有高度的保守性。通过进一步的分析比较，这类基因簇的大部分基因都具有高度的同源性，并且基因簇的分布和排列也极其相似。在灰紫红菌素的生物合成研究中，GrhO6已被证明负责催化核心5，6螺环结构的合成，而对比上述3个已知玉红霉素类基因簇中GrhO6的同源基因，发现其均具有极高的保守性。因此，我们选择rub基因簇中的RubN作为寻找β-玉红霉素产生菌的基因组挖掘的探针。
实施例2：玉红霉素类天然产物基因组挖掘分析
以RubN的氨基酸序列为探针，对NCBI数据库进行基因组挖掘，对BLAST结果进行筛选，以E值等于0进行截取，筛选到了35个同源蛋白，来源于54个测序菌株。对这54个菌株进行全基因组的antiSMASH分析，每一个菌株都能找到一个与玉红霉素类生物合成基因簇高度相似的基因簇，其中35个菌株中的基因簇表现出与grh基因簇高度同源，其余18个基因簇则多有不同之处。对这些不同菌种来源的RubN同源蛋白做进一步分析，从系统发育分析来看，菌株CB00271含有的同源蛋白和已知的RubN聚集在一簇，推测该菌株很有可能产生和玉红霉素结构类似的产物。筛选示意图如图2所示。因此我们以CB00271进行下一步的发酵验证。
实施例3：CB00271中玉红霉素基因簇分析和种属鉴定
通过antiSMASH对CB00271中的玉红霉素基因簇进行预测分析，找到了对应的玉红霉素基因簇，和已报道的β-玉红霉素生物合成基因簇rub高度相似。进一步对比分析，发现CB00271中的玉红霉素基因簇比报道的rub基因簇多出了5个修饰基因，其它基因则表现出高度相似，如图3所示。由于基因决定生物合成步骤，进而改变合成产物的结构，CB00271拥有新颖的玉红霉素基因簇，预示着其可能产生新的玉红霉素类似物。通过对已报道的β-玉红霉素产生菌进行16SrRNA进化树分析，如图4所示，可以判断CB00271是一株新的β-玉红霉素产生菌。
实施例4：链霉菌CB00271发酵分析
通过基因组挖掘，发现CB00271含有较为新颖的玉红霉素类生物合成基因簇，且与β-玉红霉素生物合成基因簇rub高度相似，即有可能产生β-玉红霉素及其新类似物。通过初步发酵，发现该菌株产生红色活性代谢产物，符合玉红霉素类化合物的性质。将CB00271发酵7天后，离心收集发酵后树脂和菌体，将树脂和菌体进行低温干燥，去除水分，用二氯甲烷和甲醇混合溶剂对树脂和菌体进行多次超声提取，直至树脂提取液无明显颜色，合并提取液，得到混合粗提液。将粗提液浓缩干燥后，将粗提物溶解在少量二氯甲烷中，并进行300-400目的硅胶柱色谱分离，用二氯甲烷和甲醇梯度洗脱，得到7个组分(Fr.1-Fr.7)；通过200目的硅胶柱依次进一步纯化Fr.3，得到5个组分(Fr.3.1-Fr.3.5)，将Fr.3.1浓缩干燥得到γ-玉红霉素；使用200目的硅胶柱进一步分离Fr3.2，得到3个组分(Fr.3.2.1-Fr.3.2.3)，将Fr.3.2.2浓缩干燥得到β-玉红霉素，其纯度达到90％以上。对其进行结构鉴定，确定为β-玉红霉素，HR-ESI-MS检测确定其分子量为[M+H]+：537.1035，分子式为C27H20O12，其数据见附图5，其核磁数据与文献报道一致。CB00271除了产生β-玉红霉素，还产生一系列类似物，根据保留时间和紫外特征吸收以及分子量，可以发现还产生少量γ-玉红霉素，3-羟基-β-玉红霉素、DK-7814-B和一些未知玉红霉素类衍生物，其中γ-玉红霉素，3-羟基-β-玉红霉素、DK-7814-B的数据见附图6-8。
实施例5：链霉菌CB00271发酵产物β-玉红霉素的分离纯化及HPLC检测
将CB00271分成加大孔树脂组和不加大孔树脂组进行7天的发酵。未加树脂组的50mL发酵液用1M HCl将发酵液pH调至3-4，混匀后，析出红褐色沉淀，将发酵液进行离心收集沉淀和菌体，用二氯甲烷和甲醇混合溶剂对离心沉淀和菌体进行多次超声提取，直至提取液无颜色，合并提取液，得到混合液，浓缩干燥后再重新定容至50mL进行HPLC检测；添加大孔树脂组离心收集发酵后树脂和菌体，用二氯甲烷和甲醇混合溶剂对树脂和菌体进行多次超声提取，直至树脂提取液无明显颜色，合并提取液，得到混合粗提液，浓缩干燥后再重新定容至50mL进行HPLC检测。HPLC分析条件及程序如下：流动相A为99.9％去离子水和0.1％甲酸；流动相B为100％乙腈，流速为1.0mL/min，紫外检测器波长为310nm，线性梯度分析程序为：0-1分钟，95％A；1-18分钟，95％A至5％A；18-20分钟，5％A；20-20.5分钟，5％A至95％A；20.5-25分钟，95％A。
实施例6：链霉菌CB00271培养基和发酵条件优化
1)采用3种碳氮源不同发酵培养基，包括G1培养基:20g/L可溶性淀粉，0.5g/LK2HPO4,0.5g/L NaCl,1g/L KNO3,0.5g/L MgSO4·7H2O,0.01g/L FeSO4·7H2O；M6培养基:60g/L蔗糖，20g/L可溶性淀粉，20g/L鱼蛋白胨,0.1g/L CuSO4·5H2O,0.005g/L NaI,2g/LCaCO3,pH 7.0；以及改良的R2A：5g/L可溶性淀粉，5g/L葡萄糖，2.5g/L蛋白胨，2.5g/L酵母粉，0.5g/L酸水解酪蛋白，0.3g/L丙酮酸钠，0.25g/L MgSO4·7H2O，0.5g/L K2HPO4，pH 7.0。每组平行3瓶，不添加树脂进行发酵，分别于发酵7天取样进行HPLC分析，评价β-玉红霉素产量。发酵结果表明，在成分复杂的改良R2A(mR2A)发酵培养基中，β-玉红霉素产量最高，产量达到40mg/L左右，具体见图9。因此CB00271自身对培养基有一定的要求和偏好，合适的培养基对β-玉红霉素产量影响大。
2)在mR2A的发酵基础上，进一步考察接种量对主产物β-玉红霉素的影响。将长好的CB00271的种子培养基，按照2％/4％/6％/10％的不同接种量分别接种至mR2A发酵培养基中，每组平行3瓶，不添加树脂进行发酵，分别于发酵7天取样进行HPLC分析，评价β-玉红霉素产量。结果表明4％的接种量β-玉红霉素的产量最高，产量达到75mg/L左右，具体结果见图10。究其原因是接种量过多会导致菌体初期生长过快，密度太大，导致菌体因营养匮乏提前死亡，缩短了代谢产物合成周期从而引起总产量降低；接种量过少会导致菌体初期密度低，生长和发酵周期过长，因此只有到达合适的接种量，才能保证在有效时间内达到正常的菌体生长密度，发挥出CB00271的最高发酵产能。
3)添加大孔吸附树脂也对β-玉红霉素产量提升有显著影响。将CB00271分成加大孔树脂组和不加大孔树脂组进行7天的发酵。未加树脂组的50mL发酵液用1M HCl将发酵液pH调至3-4，混匀后，析出红褐色沉淀，将发酵液进行离心收集沉淀和菌体，用二氯甲烷和甲醇混合溶剂对离心沉淀和菌体进行多次超声提取，直至提取液无颜色，合并提取液，得到混合液，浓缩干燥后再重新定容至50mL进行HPLC检测；添加大孔树脂组按照1％-3％的添加量在发酵培养基中添加大孔树脂，离心收集发酵后树脂和菌体，用二氯甲烷和甲醇混合溶剂对树脂和菌体进行多次超声提取，直至树脂提取液无明显颜色，合并提取液，得到混合粗提液，浓缩干燥后再重新定容至50mL进行HPLC检测，评价β-玉红霉素产量。
结果表明添加1％的时β-玉红霉素产量最高，产量达到130mg/L左右，具体结果见图11。究其原因是树脂的添加可以吸附具有细胞毒性的玉红霉素类代谢产物，消除其对菌自身的毒性，同时减少代谢产物的负反馈效应。但是树脂添加过多，会吸附培养基中的营养成分从而影响菌体的生长，导致菌体密度降低，影响产量；树脂添加过少，当树脂吸附饱和后，无法继续吸附有毒产物，负反馈调节将影响玉红霉素的生物合成，进而导致产量降低。
最终确定的最优发酵条件为：在mR2A发酵培养基中添加1％的大孔树脂，接种4％的CB00271种子进行发酵，发酵7天，β-玉红霉素产量达到最高，超过130mg/L。
上述实施例阐明的内容应当理解为这些实施例仅用于更清楚地说明本发明，而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落入本申请所附权利要求所限定的范围。
SEQUENCE LISTING
<110> 中南大学
<120> 一株链霉菌及其应用
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<170> PatentIn version 3.5
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Claims (9)
1.一株链霉菌，其特征在于，所述链霉菌为Streptomyces sp.CB00271，已于2020年6月2日保藏在中国典型培养物保藏中心，保藏编号为CCTCCNO:M 2020176。
2.如权利要求1所述的链霉菌在制备玉红霉素及其衍生物中的应用，所述玉红霉素及其衍生物的结构式为：
其中，R1和R2选自H、OH；
或
3.如权利要求2所述的应用，其特征在于，所述玉红霉素及其衍生物为β-玉红霉素，所述β-玉红霉素的结构式为：
4.如权利要求1所述链霉菌制备玉红霉素及其衍生物的方法，其特征在于，
包括以下步骤：
S1、将Streptomyces sp.CB00271发酵，后离心收集树脂和菌体，超声提取得到提取液；
S2、将提取液浓缩、干燥、分离，得到玉红霉素及其衍生物。。
5.如权利要求4所述的制备方法，其特征在于，所述超声提取用的溶剂为二氯甲烷和甲醇混合溶剂，所述为二氯甲烷和甲醇混合溶剂中二氯甲烷和甲醇按体积比1:1混合。
6.如权利要求4所述的制备方法，其特征在于，按重量份计，所述发酵的培养基的组成包括：5份可溶性淀粉，5份葡萄糖，2.5份蛋白胨，2.5份酵母粉，0.5份酸水解酪蛋白，0.3份丙酮酸钠，0.25份MgSO4·7H2O，0.5份K2HPO4，pH 5.0-7.0，并在每升培养基中添加大孔吸附树脂。
7.如权利要求4所述的制备方法，其特征在于，所述发酵的过程中，每升发酵液接种量为2％-10％；每升发酵液中添加树脂为培养基总重量的1％-3％。
8.如权利要求6所述的制备方法，其特征在于，所述大孔吸附树脂选自HP20、DA-201、XAD-8中的任意一种或几种。
9.如权利要求4所述的制备方法，其特征在于，每升发酵液中添加树脂为培养基总重量的1％-10％。
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| Rubromycins: A Class of Telomerase Inhibitor Antibiotics Produced by Streptomyces spp.;Ali Zineddine Boumehira et al.;《chapter》;20191231;全文 * |
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