WO2022247190A1 - 一种高产喜树碱内生菌株及其应用 - Google Patents

一种高产喜树碱内生菌株及其应用 Download PDF

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WO2022247190A1
WO2022247190A1 PCT/CN2021/134177 CN2021134177W WO2022247190A1 WO 2022247190 A1 WO2022247190 A1 WO 2022247190A1 CN 2021134177 W CN2021134177 W CN 2021134177W WO 2022247190 A1 WO2022247190 A1 WO 2022247190A1
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camptothecin
zcmukl
preservation
alternaria
strain
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开国银
冯岳
黄超
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浙江中医药大学
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    • C—CHEMISTRY; METALLURGY
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus

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  • the invention relates to an endophytic fungus capable of high camptothecin production, which belongs to the research field of medicinal plant endophytes.
  • Camptothecin is a monoterpene indole alkaloid with unique topoisomerase I inhibition and thus unique inhibitory activity against cancer.
  • camptothecin anti-tumor drugs with good drug efficacy, low toxicity and side effects and good solubility have been screened out and used in clinical practice. It is the third largest plant anti-cancer drug.
  • camptothecin is the main source of camptothecin production, but its content is still low, and it is very difficult to directly extract from camptothecin, which can no longer meet the requirements. Great demand in the medical market. Therefore, it is of great significance to obtain alternative sources for the production of camptothecin.
  • Endophytes are a group of filamentous fungi that live in plant tissues. During the long-term evolution process, some endophytes have established a mutualistic symbiotic relationship with their host plants: endophytes absorb nutrients from plants, and their secondary metabolites can regulate the plant's immune system, promote plant growth, and increase plant resistance to diseases. Or tolerance to abiotic stresses such as temperature, pH, and heavy metals. At the same time, there may be an exchange of genetic material between the endophyte and the plant, and the endophyte can partially or completely synthesize the same active ingredients as the host.
  • camptothecin because microorganisms such as endophytes have the characteristics of low culture cost, rapid growth, and short fermentation cycle, the endophyte isolated from camptothecin is expected to become a potential source of high-yield camptothecin.
  • the primary problem to be solved by the present invention is to provide an endophytic fungus capable of efficiently producing camptothecin.
  • the strain was isolated from the seeds of Campylodon chinensis. After isolation and identification, it was confirmed that the high-yielding camptothecin endophytic strain is:
  • Alternaria temuissima ZCMUKL-S1 (Alternaria temuissima ZCMUKL-S1), its preservation number is CCTCC NO: M2021189, the preservation date is February 23, 2021, and the preservation unit is the China Center for Type Culture Collection.
  • the preservation number is CCTCC NO: M2021190
  • the preservation date is February 23, 2021
  • the preservation unit is the China Center for Type Culture Collection.
  • the preservation number is CCTCC NO: M2021191
  • the preservation date is February 23, 2021
  • the preservation unit is the China Center for Type Culture Collection.
  • the present invention also provides the application of the high camptothecin-producing endophytic strain in preparing camptothecin.
  • camptothecin is specifically realized through the following steps:
  • the concentration of the spore suspension is 6.0 ⁇ 10 6 spores/mL.
  • the volume ratio of the spore suspension to the potato dextrose medium is 1:100.
  • potato glucose medium is: potato 200g/L, glucose 20g/L, agar 15g/L.
  • the culture condition is 25° C., 150 rpm, and the culture time is 7 days.
  • the present invention provides an endophytic fungus capable of high camptothecin production, and the endophytic fungus has a higher yield of camptothecin, wherein, the unit dry mycelia of Aspergillus versicolor ZCMUKL-S3
  • the camptothecin yield of the powder is as high as 116 ⁇ g/g, which is the highest yield of camptothecin produced by endophytic bacteria so far, and has potential application value for its industrial production.
  • the endophytic fungus capable of high camptothecin production provided by the present invention has good genetic stability, and can still maintain 80% of the initial yield after 10 generations of subculture.
  • Fig. 1 is the colony form diagram of camptothecin-producing endophytic bacteria, wherein, a is the S1 strain, b is the S2 strain, and c is the S3 strain;
  • Fig. 2 is the electron micrograph of camptothecin-producing endophytic bacteria under different magnifications, wherein, a is the S1 strain, b is the S2 strain, and c is the S3 strain;
  • Fig. 3 is the evolutionary relationship diagram of camptothecin-producing endophytes S1, S2 and S3;
  • Fig. 4 is a graph showing the results of HPLC detection of camptothecin content of endophytic bacteria of each generation, wherein a is the S1 strain, b is the S2 strain, and c is the S3 strain.
  • Embodiment 1 the separation of endophytic fungus of camphor tree seed
  • the morphology of the S1 colony is as follows: the primary hyphae are white, and the aerial hyphae are more developed. As the culture time prolongs, the center of the colony becomes dark green, the opposite side of the colony turns brownish yellow, the texture of the colony is fluffy, and the edges of the colony are irregular , no exudate.
  • the colony morphology of S2 is as follows: the primary hyphae are white, the colony texture is fluffy, and the center of the colony is black.
  • S3 colony is as follows: at the initial stage of culture, it is white and round, with smooth edges, dry surface, and grows rapidly.
  • the colony is radially grooved, surrounded by white hyphae in the middle, and is gray-green in color. After 4-5 days, the back of the colony turns yellow ( Figure 1).
  • Embodiment 3 endophytic fungus S1, S2, S3 microscopic examination and identification of producing camptothecin
  • the isolated strains were stained with lactophenol cotton blue, and their morphology was observed under a microscope. Microscopic analysis shows in Figure 2 that the vegetative mycelium of the S1 strain is composed of branched hyphae with transverse septa, and the spores are relatively thin. , Rarely seen, with obvious diaphragm and mediastinum.
  • the hyphae of the S2 strain were filamentous and transparent. Conidia are few and mostly solitary.
  • the conidia of S3 strain had fewer branches, the ends were radial, the top was nearly spherical, and green spores were produced. The spores were ovoid.
  • the conidia have smooth walls and hyphae are transparent.
  • Embodiment 4 endophytic fungus molecular biology identification of producing camptothecin
  • Genomic DNA of fungi was extracted by CTAB method and classified according to its 18s sequence. Using p1 (5'-GTAGTCATATGCTTGTCTC-3') and p2 (5'-TCCGCAGGTTCACCTACGGA-3') as primers, the 18s sequence was amplified using genomic DNA as a template.
  • the PCR reaction conditions were: 94° C. for 5 min, 30 cycles (98° C. for 15 s, 55° C. for 15 s, 72° C. for 120 s), and 72° C. for 10 min.
  • PCR amplification system template 1 ⁇ L, upstream and downstream primers p1 and p2 1 ⁇ L each, dNTP Mix 4 ⁇ L, 5 ⁇ primeSTAR Buffer 10 ⁇ L, sterilized double distilled water 32.5 ⁇ L, primeSTAR DNA polymerase 0.5 ⁇ L.
  • PCR products were purified and recovered using a gel recovery kit. Sequencing compared the sequences with those in the GenBank database. Phylogenetic tree analysis results are shown in Figure 3, the 18s sequence of S1 is highly similar to Alternaria tenuissima; the 18s sequence of S2 is similar to Alternaria citricancri; the 18s sequence of S3 is similar to Aspergillus versicolor (Aspergillus versicolor) similar.
  • the endophytic fungus S1 of the present invention is classified as Alternaria temuissima ZCMUKL-S1 (Alternaria temuissima ZCMUKL-S1), the preservation number is CCTCC NO: M2021189, the preservation date is February 23, 2021, and the preservation unit is China
  • the Center for Type Culture Collection (CCTCC) is located at Wuhan University, Wuhan, China, postcode 430072.
  • the endophytic fungus S2 of the present invention is classified as Alternaria citricancri ZCMUKL-S2 (Alternaria citricancri ZCMUKL-S2), the preservation number is CCTCC NO: M2021190, the preservation date is February 23, 2021, and the preservation unit is China
  • the Center for Type Culture Collection (CCTCC) is located at Wuhan University, Wuhan, China, postcode 430072.
  • the endophytic fungus S3 of the present invention is classified as Aspergillus versicolor ZCMUKL-S3 (Aspergillus versicolor ZCMUKL-S3), the preservation number is CCTCC NO: M2021191, the preservation date is February 23, 2021, and the preservation unit is Chinese typical culture
  • the depository center (CCTCC) is located at Wuhan University, Wuhan, China, postcode 430072.
  • Embodiment 5 endophytic fungus culture
  • the fungal spores of S1, S2, and S3 grown on the PDA medium were washed with 50 mL of sterile saline, respectively, and filtered through sterile gauze to prepare S1, S2, and S3 spore suspensions. Inoculate 1 mL of spore suspension (6.0 ⁇ 10 6 spores/mL) into 100 mL of PDA liquid medium for camptothecin production. Cultivate at 25°C and 150rpm for 7 days, collect mycelium by centrifugation at 9000 ⁇ g for 5 min, freeze-dry and measure its dry mass, and measure its camptothecin content by HPLC.
  • Embodiment 6 detection of endophytic fungus camptothecin output
  • the mycelium cultured for 7 days was collected by centrifugation, and dried in an oven at 60°C to constant weight.
  • the supernatant was collected by centrifugation, dried by rotary evaporation, and redissolved in 1 mL of DMSO and methanol (volume ratio 1:50), and the content of camptothecin in endophyte mycelium was detected by HPLC.
  • S1 can produce about 2.3 ⁇ g/g camptothecin
  • S2 can produce about 242 ng/g camptothecin
  • S3 can produce about 116 ⁇ g/g camptothecin.
  • the yield of S3 is the highest yield of camptothecin produced by endophytic bacteria, which has potential application value in its industrial production.
  • the above S1, S2, and S3 strains were inoculated into PDA solid medium, and successively subcultured for 10 generations respectively.
  • the growth performance and morphology of the strains were observed, and it was found that there was no significant difference in growth performance and morphology among the strains of each generation.
  • the strains of each generation were successively inoculated into PDA liquid medium for fermentation and culture, and the content of camptothecin in each generation of strains was detected, and the content of camptothecin was only slightly reduced, which indicated that the S1, S2, and S3 strains had better genetic Stability ( FIG. 4 ), wherein, the S1 strain can still maintain more than 65% of the initial yield after 10 generations of passage, and the S3 strain can still maintain about 80% of the initial yield after 10 generations of passage. .

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Abstract

提供了一种高产喜树碱内生菌株，其选自保藏编号为CCTCC NO：M2021189的细极链格孢菌（Alternaria tenuissima）ZCMUKL-S1，或保藏编号为CCTCC NO：M2021190的香茅链格孢菌（Alternaria citricancri）ZCMUKL-S2，或保藏编号为CCTCC NO：M2021191的杂色曲霉（Aspergillus versicolor）ZCMUKL-S3。该内生菌株具有较高的喜树碱产量，其中该杂色曲霉ZCMUKL-S3的单位干燥菌丝体粉末的喜树碱产量高达116μg/g。

Description

一种高产喜树碱内生菌株及其应用 技术领域
本发明涉及一株可高产喜树碱的内生真菌，属于药用植物内生菌研究领域。
技术背景
喜树碱是一种单萜吲哚生物碱，具有独特的拓扑异构酶Ⅰ抑制作用，因此对癌症具有独特的抑制活性。多种药效好、毒副作用小和溶解性能好的喜树碱类抗肿瘤新药已被筛选出来并应用于临床，是第三大植物抗癌药物。
喜树、蛇根草等药用植物均可生物合成喜树碱，其中喜树是喜树碱生产的主要来源，但其含量仍较低，且从喜树中直接提取十分困难，已不能满足医疗市场极大的需求。因此，获得生产喜树碱的替代来源具有重要意义。
内生真菌是一类寄生在植物组织内的丝状真菌。在长期进化过程中，一些内生菌与其宿主植物间建立了互惠共生关系：内生菌从植物中吸取营养，其次级代谢产物又可调节植物的免疫系统、促进植物的生长、增加植物对病害或温度、pH、重金属等非生物胁迫的耐受性。同时，内生菌与植物之间可能伴随发生着遗传物质的交换，内生菌能部分或全部合成与宿主相同的活性成分。此外，由于内生菌等微生物具有培养成本低，生长迅速，发酵周期短等特点，因此，从喜树中分离获得的内生真菌有望成为高产喜树碱的潜在来源。
在内生菌生产药用成分的过程中，遗传稳定性差等特点限制了其工业化应用，因此，获得可稳定遗传的喜树碱内生菌生产菌株具有重要意义。
发明内容
本发明首要解决的问题是提供了一种可高效生产喜树碱的内生真菌。菌株分离自喜树种子。经分离鉴定，确认所述高产喜树碱内生菌株为：
细极链格孢菌ZCMUKL-S1(Alternaria temuissima ZCMUKL-S1)，其保藏编号为CCTCC NO：M2021189，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心。
或香茅链格孢菌ZCMUKL-S2(Alternaria citricancri ZCMUKL-S2)，保藏编号为CCTCC NO：M2021190，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心。
或杂色曲霉ZCMUKL-S3(Aspergillus versicolor ZCMUKL-S3)，保藏编号为CCTCC NO：M2021191，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心。
本发明还提供了所述高产喜树碱内生菌株在制备喜树碱中的应用。
进一步地，制备喜树碱具体通过如下步骤实现：
(1)挑取高产喜树碱内生菌株的真菌孢子，用无菌生理盐水洗涤、过滤，制成孢子悬浮液。
(2)将孢子悬浮液接种到马铃薯葡萄糖琼脂培养基中培养，然后收集上清液并分离获得喜树碱。
进一步地，所述步骤1中，孢子悬浮液的浓度为6.0×10 6孢子/mL。
进一步地，所述步骤1中，孢子悬浮液与马铃薯葡萄糖培养基的体积比为1:100。
进一步地，马铃薯葡萄糖培养基的配方为：马铃薯200g/L，葡萄糖20g/L，琼脂15g/L。
进一步地，所述步骤2中，培养条件为25℃，150rpm，培养时间为7天。
本发明的有益效果是：本发明提供了一种可高产喜树碱的内生真菌，该内生真菌具有较高的喜树碱产量，其中，杂色曲霉ZCMUKL-S3的单位干燥菌丝体粉末的喜树碱产量高达116μg/g，是目前已报道的内生菌产喜树碱的最高产量，对其工业化生产具有潜在的应用价值。另外，本发明提供的可高产喜树碱的内生真菌具有较好的遗传稳定性，10代传代后仍能保持初始产量的80％。
附图说明
图1为产喜树碱内生菌的菌落形态图，其中，a为S1菌株，b为S2菌株，c为S3菌株；
图2为产喜树碱内生菌不同放大倍数下的电镜图，其中，a为S1菌株，b为S2菌株，c为S3菌株；
图3为产喜树碱内生菌S1、S2和S3的进化关系图；
图4为HPLC检测各代内生菌喜树碱含量结果图，其中，a为S1菌株，b为S2菌株，c为S3菌株。
具体实施方式
实施例1、喜树种子内生真菌分离
首先，用自来水过夜冲洗喜树种子。然后用75％(v/v)乙醇清洗种子3分钟，无菌水冲洗5次，50％(v/v)次氯酸钠消毒5分钟，无菌水再次冲洗5次进行表面消毒，最后在无菌滤纸上干燥。随后，将表面已灭菌的喜树种子切割至2mm大小，并置于PDA培养基上，25℃培养5-10天，定期纯化获得S1、S2、S3三个单菌落。
实施例2、S1、S2、S3单菌落的菌落形态分析
利用形态学方法鉴定内生真菌。
如图1所示，S1菌落形态如下：初生菌丝为白色，气生菌丝较发达，随培养时间延长菌落中央呈暗绿色，菌落反面呈棕黄色，菌落质地呈绒毛状，菌落边缘不整齐，无渗出液。
S2菌落形态如下：初生菌丝为白色，菌落质地呈绒毛状，菌落中央呈黑色。
S3菌落形态如下：培养初期，白色圆形，边缘光滑，表面干燥，生长迅速。菌落呈放射状凹槽，中间被白色菌丝环绕，呈灰绿色。4-5天后，菌落背面呈现黄色(图1)。
实施例3、产喜树碱的内生真菌S1、S2、S3显微镜检鉴定
分离的菌株用乳酚棉蓝染色，并于显微镜下观察形态。显微分析如图2显示，S1菌株营养菌丝由具横隔的分支菌丝构成，歯丝较细，分生孢子梗从菌丝生出，直立、短小，顶端形成分生孢子，多单生，鲜见唯生，具明显的横隔和纵隔。S2菌株菌丝呈现丝状，菌丝透明。分生孢子较少，多单生。S3菌株分生孢子分枝较少，末端呈放射状，顶部近球形，产生绿色孢子，孢子卵球形。分生孢子壁光滑，菌丝透明。
实施例4、产喜树碱的内生真菌分子生物学鉴定
采用CTAB法提取真菌基因组DNA，根据其18s序列进行分类。以p1(5'-GTAGTCATATGCTTGTCTC-3')、p2(5'-TCCGCAGGTTCACCTACGGA-3')为引物，以基因组DNA为模板扩增其18s序列。PCR反应条件为：94℃5min，30个循环(98℃15s、55℃15s、72℃120s)，72℃10min。PCR扩增体系：模板1μL，上下游引物p1、p2各1μL，dNTP Mix 4μL，5×primeSTAR Buffer 10μL，灭菌的双蒸水32.5μL，primeSTAR DNA聚合酶0.5μL。采用胶回收试剂盒对PCR产物进行纯化和回收。经测序将序列与GenBank数据库中的序列进行比较。进化树分析结果如图3显示，S1的18s序列与细极链格孢菌(Alternaria tenuissima)高度相似；S2的18s序列与香茅链格孢菌Alternaria citricancri相似；S3的18s序列与杂色曲霉(Aspergillus versicolor)相似。本发明的内生真菌S1，分类命名为细极链格孢菌ZCMUKL-S1(Alternaria temuissima ZCMUKL-S1)，保藏编号为CCTCC NO：M2021189，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心(CCTCC)，地址为中国.武汉.武汉大学，邮编为430072。本发明的内生真菌S2，分类命名为香茅链格孢菌ZCMUKL-S2(Alternaria citricancri ZCMUKL-S2)，保藏编号为CCTCC NO：M2021190，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心(CCTCC)，地址为中国.武汉.武汉大学，邮编为430072。本发明的内生真菌S3，分类命名为杂色曲霉ZCMUKL-S3(Aspergillus versicolor ZCMUKL-S3)，保藏编号为CCTCC NO：M2021191，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心(CCTCC)，地址为中国.武汉.武汉大学，邮编为430072。
实施例5、内生真菌培养
将PDA培养基上生长的S1、S2、S3的真菌孢子分别用50mL无菌生理盐水洗涤，用无菌纱布过滤，制备S1、S2、S3孢子悬浮液。将1mL孢子悬浮液(6.0×10 6孢子/mL)接种到100mL PDA液体培养基中，用于喜树碱的生产。在25℃，150rpm的条件下培养7天，9000×g 5 min离心收集菌丝体，冻干后测定其干质量，用HPLC法测其喜树碱含量。
实施例6、内生真菌喜树碱产量检测
(1)为检测菌丝体中的喜树碱，离心收集培养7天的菌丝体，于60℃烘箱中干燥至恒重。称取0.3g干燥菌丝体粉末并重悬于40ml二氯甲烷和甲醇(体积比4:1)溶液中，超声提取10min，得到均匀的悬浮液。离心收集上清液，经旋转蒸发干燥后复溶于1mL的DMSO与甲醇(体积比1:50)的溶液中，采用HPLC检测内生菌菌丝体喜树碱含量。
(2)HPLC色谱柱规格：C-18反相硅胶柱不锈钢柱，ZORBAX SB-C18，粒径5μm，规格4.6×150mm；进样量：10μL；柱温：30℃；流速：1mL/min；(2)流动相及检测波长：喜树碱流动相：色谱纯乙腈：水＝35：65(V：V)；检测波长：254nm。
经分析检测S1能产生约2.3μg/g的喜树碱，S2能产生约242ng/g的喜树碱，S3能产生约116μg/g的喜树碱。其中，S3的产量是目前已报道的内生菌产喜树碱的最高产量，对其工业化生产具有潜在的应用价值。
实施例7、产喜树碱内生菌S3遗传稳定性评价
将上述S1、S2、S3菌株接种至PDA固体培养基，并分别连续继代培养10代，观察菌株生长性能及形态发现，其各代菌株之间其生长性能与形态均无明显差异。随后，依次将各代菌株接种至PDA液体培养基进行发酵培养，检测各代菌株喜树碱含量发现，其喜树碱含量仅轻微的降低，这表明S1、S2、S3菌株具有较好的遗传稳定性(图4)，其中，S1菌株10代传代后仍能保持初始产量的65％以上，S3菌株10代传代后仍能保持初始产量约80％。。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (7)

  1. 一种高产喜树碱内生菌株，其特征在于，所述高产喜树碱内生菌株为：
    细极链格孢菌ZCMUKL-S1(Alternaria temuissima ZCMUKL-S1)，其保藏编号为CCTCC NO：M2021189，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心。
    或香茅链格孢菌ZCMUKL-S2(Alternaria citricancri ZCMUKL-S2)，保藏编号为CCTCC NO：M2021190，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心。
    或杂色曲霉ZCMUKL-S3(Aspergillus versicolor ZCMUKL-S3)，保藏编号为CCTCC NO：M2021191，保藏日期为2021年2月23日，保藏单位为中国典型培养物保藏中心。
  2. 一种权利要求1所述高产喜树碱内生菌株在制备喜树碱中的应用。
  3. 根据权利要求2所述的应用，其特征在于，制备喜树碱具体通过如下步骤实现：
    (1)挑取高产喜树碱内生菌株的真菌孢子，用无菌生理盐水洗涤、过滤，制成孢子悬浮液。
    (2)将孢子悬浮液接种到马铃薯葡萄糖琼脂培养基中培养，然后收集上清液并分离获得喜树碱。
  4. 根据权利要求3所述的应用，其特征在于，所述步骤1中，孢子悬浮液的浓度为6.0×10 6孢子/mL。
  5. 根据权利要求4所述的应用，其特征在于，所述步骤1中，孢子悬浮液与马铃薯葡萄糖培养基的体积比为1:100。
  6. 根据权利要求3所述的应用，其特征在于，马铃薯葡萄糖培养基的配方为：马铃薯200g/L，葡萄糖20g/L，琼脂15g/L。
  7. 根据权利要求3所述的应用，其特征在于，所述步骤2中，培养条件为25℃，150rpm，培养时间为7天。
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