微生物学报

Cluster	Position of gene cluster (start)	Position of gene cluster (termination)	Type of gene cluster	Most similar known cluster	Similarity (%)
−: No similar gene cluster predicted.
1	6 891	68 544	T3PKS, NRPS, T1PKS	Alkylresorcinol	100
2	229 260	250 820	Melanin, terpene	Melanin	57
3	643 925	689 547	NRPS-like, T1PKS	Olimycin A/B	20
4	1 188 455	1 208 783	Terpene	Clipibycyclene	6
5	1 362 768	1 402 798	T3PKS	Flaviolin/1,3,6,8-tetrahydro xynaphthalene	100
6	1 921 470	1 953 655	NAPAA	ε-poly-L-lysine	100
7	2 176 155	2 186 553	Ectoine	Ectoine	100
8	2 704 623	2 753 867	T1PKS, NRPS	Rifamorpholine A/B/C/D/E	3
9	2 968 910	2 989 421	Terpene	SCO-2138	14
10	3 256 213	3 291 146	NAPAA	Stenothricin	13
11	3 352 468	3 363 133	Melanin	Melanin	40
12	3 457 262	3 468 035	NI-siderophore	Desferrioxamin B/E	83
13	4 717 147	4 728 088	Butyrolactone	Coelimycin P1	8
14	5 185 780	5 258 286	T2PKS, Oligosaccharide, PKS-like	Cytorhodin	84
15	5 792 861	5 803 409	Butyrolactone	−	−
16	5 813 805	5 824 746	Butyrolactone	Neocarzinostatin	4
17	5 900 336	5 941 140	2dos	Notonesomycin A	6
18	6 189 388	6 209 988	Terpene	Albaflavenone	100
19	6 843 605	6 854 598	NI-siderophore	−	−
20	7 142 887	7 153 846	RiPP-like	−	−
21	7 217 971	7 238 601	Terpene	Geosmin	100
22	7 388 394	7 411 573	RRE-containing	−	−
23	7 481 332	7 619 277	NI-siderophore, NRPS, NRPS-like	Friulimicin A/B/C/D	33
24	8 025 082	8 052 261	Terpene	Hopene	92
25	8 199 733	8 258 068	NRP-metallophore, NRPS	Coelichelin	100
26	8 393 130	8 434 467	Ladderane	Colabomycin	15
27	8 465 947	8 507 131	T3PKS	Germicidin	100
28	8 572 827	8 586 380	NI-siderophore	Peucechelin	25
29	8 711 211	8 738 249	RiPP-like, lanthipeptide-class-iii	Informatipeptin	100
30	8 800 286	8 844 260	T1PKS	Chlorothricin/Deschlorothricin	4
31	8 914 379	8 958 647	NRPS	Diisonitrile antibiotic SF2768	55

Cluster	Position of gene cluster (start)	Position of gene cluster (termination)	Type of gene cluster	Most similar known cluster	Similarity (%)
−: No similar gene cluster predicted.
1	6 891	68 544	T3PKS, NRPS, T1PKS	Alkylresorcinol	100
2	229 260	250 820	Melanin, terpene	Melanin	57
3	643 925	689 547	NRPS-like, T1PKS	Olimycin A/B	20
4	1 188 455	1 208 783	Terpene	Clipibycyclene	6
5	1 362 768	1 402 798	T3PKS	Flaviolin/1,3,6,8-tetrahydro xynaphthalene	100
6	1 921 470	1 953 655	NAPAA	ε-poly-L-lysine	100
7	2 176 155	2 186 553	Ectoine	Ectoine	100
8	2 704 623	2 753 867	T1PKS, NRPS	Rifamorpholine A/B/C/D/E	3
9	2 968 910	2 989 421	Terpene	SCO-2138	14
10	3 256 213	3 291 146	NAPAA	Stenothricin	13
11	3 352 468	3 363 133	Melanin	Melanin	40
12	3 457 262	3 468 035	NI-siderophore	Desferrioxamin B/E	83
13	4 717 147	4 728 088	Butyrolactone	Coelimycin P1	8
14	5 185 780	5 258 286	T2PKS, Oligosaccharide, PKS-like	Cytorhodin	84
15	5 792 861	5 803 409	Butyrolactone	−	−
16	5 813 805	5 824 746	Butyrolactone	Neocarzinostatin	4
17	5 900 336	5 941 140	2dos	Notonesomycin A	6
18	6 189 388	6 209 988	Terpene	Albaflavenone	100
19	6 843 605	6 854 598	NI-siderophore	−	−
20	7 142 887	7 153 846	RiPP-like	−	−
21	7 217 971	7 238 601	Terpene	Geosmin	100
22	7 388 394	7 411 573	RRE-containing	−	−
23	7 481 332	7 619 277	NI-siderophore, NRPS, NRPS-like	Friulimicin A/B/C/D	33
24	8 025 082	8 052 261	Terpene	Hopene	92
25	8 199 733	8 258 068	NRP-metallophore, NRPS	Coelichelin	100
26	8 393 130	8 434 467	Ladderane	Colabomycin	15
27	8 465 947	8 507 131	T3PKS	Germicidin	100
28	8 572 827	8 586 380	NI-siderophore	Peucechelin	25
29	8 711 211	8 738 249	RiPP-like, lanthipeptide-class-iii	Informatipeptin	100
30	8 800 286	8 844 260	T1PKS	Chlorothricin/Deschlorothricin	4
31	8 914 379	8 958 647	NRPS	Diisonitrile antibiotic SF2768	55

Compound 1 (ε-rhodomycinone)		Compound 2 (η-rhodomycinone)
1	119.34, CH	7.79, d (9.4)	157.84, C
2	139.90, CH	7.75, d (7.0)	130.73, CH	7.39, m	4
3	124.91, CH	7.37, d (7.7)	129.81, CH	7.39, m
4	162.16, C		157.88, C
4a	116.82, C		113.27, C
5	189.77, C		186.35, C
5a	111.22, C		131.64, C
6	156.87, C		119.69, CH	7.93, s	7, 11a, 10a, 5
6a	134.35, C		149.23, C
7	61.39, CH	5.55, m	65.95, CH	4.84, t (4.5)
8	32.71, CH₂	2.06, m	37.89, CH₂	Hα 2.18, m	6a, 7, 9
				Hβ 1.98, m
9	71.51, C		71.46, C
10	51.82, CH	4.15, s	51.84, CH	4.12, s	8, 9, 10a, 6a, 11, 15
10a	137.51, C		130.04, C
11	157.80, C		161.36, C
11a	111.52, C		114.42, C
12	185.87, C		189.53, C
12a	133.53, C		113.11, C
13	35.22, CH₂	Hα 1.70, m	32.20 CH₂	Hα 1.62, m	14, 8, 10, 9
		Hβ 1.45, m		Hβ 1.48, m
14	7.29, CH₃	1.05, t (7.3)	7.45, CH₃	1.03, t (7.2)	13, 9
15	171.20, C		171.72, C
16	52.71, CH₃	3.65, s	52.44, CH₃	3.64, s	15

Compound 1 (ε-rhodomycinone)		Compound 2 (η-rhodomycinone)
1	119.34, CH	7.79, d (9.4)	157.84, C
2	139.90, CH	7.75, d (7.0)	130.73, CH	7.39, m	4
3	124.91, CH	7.37, d (7.7)	129.81, CH	7.39, m
4	162.16, C		157.88, C
4a	116.82, C		113.27, C
5	189.77, C		186.35, C
5a	111.22, C		131.64, C
6	156.87, C		119.69, CH	7.93, s	7, 11a, 10a, 5
6a	134.35, C		149.23, C
7	61.39, CH	5.55, m	65.95, CH	4.84, t (4.5)
8	32.71, CH₂	2.06, m	37.89, CH₂	Hα 2.18, m	6a, 7, 9
				Hβ 1.98, m
9	71.51, C		71.46, C
10	51.82, CH	4.15, s	51.84, CH	4.12, s	8, 9, 10a, 6a, 11, 15
10a	137.51, C		130.04, C
11	157.80, C		161.36, C
11a	111.52, C		114.42, C
12	185.87, C		189.53, C
12a	133.53, C		113.11, C
13	35.22, CH₂	Hα 1.70, m	32.20 CH₂	Hα 1.62, m	14, 8, 10, 9
		Hβ 1.45, m		Hβ 1.48, m
14	7.29, CH₃	1.05, t (7.3)	7.45, CH₃	1.03, t (7.2)	13, 9
15	171.20, C		171.72, C
16	52.71, CH₃	3.65, s	52.44, CH₃	3.64, s	15

青风藤内生链霉菌SZC001生物合成潜力分析及抗肿瘤天然产物η-rhodomycinone的鉴定

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秦雨晴 ^# , 田程 ^# , 杨浩 , 蒲洪 , 漆宇昕 , 邹玮 , 江丹丹 , 黄雪霜 ^* , 孙润泽 ^*

微生物学报 | 研究报告 2024,64(12): 4804-4816

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微生物学报 | 研究报告 2024, 64(12): 4804-4816

青风藤内生链霉菌SZC001生物合成潜力分析及抗肿瘤天然产物η-rhodomycinone的鉴定

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秦雨晴^#, 田程^#, 杨浩, 蒲洪, 漆宇昕, 邹玮, 江丹丹, 黄雪霜^*, 孙润泽^*

作者信息

湖南医药学院, 中药合成生物学研究湖南省重点实验室, 湖南怀化 418000

Biosynthetic potential and anti-tumor natural product η-rhodomycinone of the endophyte Streptomyces sp. SZC001 in Sinomenium acutum

Yuqing QIN^#, Cheng TIAN^#, Hao YANG, Hong PU, Yuxin QI, Wei ZOU, Dandan JIANG, Xueshuang HUANG^*, Runze SUN^*

Affiliations

Hunan Provincial Key Laboratory for Synthetic Biology of Traditional Chinese Medicine, Hunan University of Medicine, Huaihua 418000, Hunan, China

出版时间: 2024-09-27 doi: 10.13343/j.cnki.wsxb.20240370

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摘要

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【目的】探究青风藤内生链霉菌SZC001的次级代谢产物生物合成潜力，挖掘未知的活性天然产物。【方法】表面消毒法分离SZC001并利用三代联合二代测序获得完整基因组，结合antiSMASH分析评估其天然产物合成潜力；使用4种培养基发酵初筛代谢谱，通过硅胶柱层析、高效液相制备、高分辨质谱以及核磁共振仪等对化合物进行分离鉴定；利用CCK-8法进行目标化合物的细胞毒性测定。【结果】鉴定研究菌株为链霉菌SZC001，基因组总长度9 109 166 bp，G+C含量71.08%；antiSMASH显示该菌株共含有31个潜在的生物合成基因簇，有17个基因簇与已知基因簇的相似度小于40%；在4种培养基中可产生多个蒽环类天然产物，经分离鉴定得到含量最高的2个化合物1和2。化合物1为已知化合物ε-rhodomycinone；化合物2为一个新化合物η-rhodomycinone，相对已知化合物α₂-rhodomycinone其在C-10位羟基衍生化并且骨架6位羟基变为11位。化合物1与2对2种肿瘤细胞系均有较好的抑制活性，半抑制浓度(half maximal inhibitory concentration, IC₅₀)为1.55−4.59 μmol/L。【结论】SZC001是一株有挖掘活性天然产物潜力的菌株，从中获得的蒽醌类天然产物具有较好的抗肿瘤活性，后续值得进一步开发和利用。本研究增加了ε-rhodomycinone衍生物的种类，为青风藤内生菌资源的后续开发提供了基础。

关键词

青风藤 / 内生链霉菌 / 天然产物 / 蒽环类化合物 / η-rhodomycinone

Abstract

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[Objective] To investigate the biosynthetic potential of the endophyte Streptomyces sp. SZC001 in Sinomenium acutum and explore the unknown active natural products. [Methods] SZC001 was isolated by surface sterilizing method and its full-length genome sequence was obtained by third-generation and second-generation sequencing. Then, the biosynthetic potential of SZC001 was evaluated by antiSMASH analysis. Fermentation was carried out with four media, and the compounds were separated and identified by silica gel column chromatography, high-performance liquid chromatography, high-resolution mass spectrometry and nuclear magnetic resonance. The CCK-8 assay was employed to examine the cytotoxicity of the target compounds. [Results] The strain was identified as Streptomyces sp. SZC001, with a genome length of 9 109 166 bp and the G+C content of 71.08%. The antiSMASH analysis showed that the genome of the strain contained a total of 31 biosynthetic gene clusters (BGCs), among which 17 BGCs had less than 40% similarity with the known BGCs. The strain produced several anthracyclines in four media, and the two most abundant compounds 1 and 2 were isolated and identified. Compound 1 is the known compound ε-rhodomycinone and compound 2 is a new compound η-rhodomycinoe, which is derived from α₂-rhodomycinone by derivatization of the hydroxyl group at the C-10 position and transfer of the hydroxyl group at the 6-position to the 11-position of the backbone. Both compounds 1 and 2 showed relatively strong inhibitory effects on two tumor cell lines, with IC₅₀ of 1.55–4.59 μmol/L. [Conclusion] SZC001 is a strain that holds the potential for the exploration of active natural products. The anthracyclines produced by the strain exhibit anti-tumor activities and warrant further development and utilization. This study enriches ε-rhodomycinone derivatives, furnishing a foundation for the subsequent exploitation of the endophytic resources of S. acutum.

Key words

Sinomenium acutum / endophytic Streptomyces / natural product / anthracyclines / η-rhodomycinone

引用本文

秦雨晴, 田程, 杨浩, 蒲洪, 漆宇昕, 邹玮, 江丹丹, 黄雪霜, 孙润泽. 青风藤内生链霉菌SZC001生物合成潜力分析及抗肿瘤天然产物η-rhodomycinone的鉴定. 微生物学报, 2024 , 64 (12) : 4804 -4816 . DOI: 10.13343/j.cnki.wsxb.20240370

Yuqing QIN, Cheng TIAN, Hao YANG, Hong PU, Yuxin QI, Wei ZOU, Dandan JIANG, Xueshuang HUANG, Runze SUN. Biosynthetic potential and anti-tumor natural product η-rhodomycinone of the endophyte Streptomyces sp. SZC001 in Sinomenium acutum[J]. Acta Microbiologica Sinica, 2024 , 64 (12) : 4804 -4816 . DOI: 10.13343/j.cnki.wsxb.20240370

正文

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植物内生菌是指存在于植物内部且暂不致病的一类微生物，包括内生真菌与内生细菌等^[1-2]。植物内生菌是挖掘新的微生物菌种及新结构活性天然产物的重要来源^[3-4]。近年来，从中药植物铁皮石斛(Dendrobium officinale)、直立百部(Stemona sessilifolia)、丹参(Salvia miltiorrhiza)、矮小扁枝石松(Diphasiastrum veitchii)中均得到了验证^[5-8]。青风藤(Sinomenium acutum)为治疗风湿痹病的传统中药，其有效成分青藤碱(sinomenine, SIN)可治疗类风湿关节炎、慢性肾炎与痛风性关节炎等，具有重要的经济价值^[9-11]。然而，青风藤的微生物资源尚待深入发掘，当前关于其内生菌的研究文献较少。例如2015年肖健等^[12]对青风藤内生真菌QT-NJ-10研究得到了一个已知化合物eremofortine C-1，后续再无其他报道，其内生细菌研究基本处于空白。

紫红霉酮(ε-rhodomycinone)于1985年从链霉菌(Streptomyces. sp) HPL Y-11472中分离得到^[13]。它与一线抗肿瘤药物柔红霉素及阿霉素具有相似的骨架，但在糖基及羟基等基团的修饰存在差异(图1)^[14]，这些差异导致它们的抗肿瘤活性及细胞毒性各异^[15]。ε-rhodomycinone对人肺癌细胞、人乳腺癌细胞、人肝癌细胞及人结肠癌细胞具有较好的抑制活性，相对于阿霉素及柔红霉素具有更低的心脏毒性^[16-17]。此外它还具有明显的抗疟疾活性，对疟原虫(Plasmodium. falciparum) K-1的半抑制浓度(half maximal inhibitory concentration, IC₅₀)为8.88 μg/mL^[18]。ε-rhodomycinone是柔红霉素、阿霉素、cytorhodin等抗肿瘤化合物重要的生物合成前体^[19-23]。此外在阿霉素的发现菌株如波赛链霉菌(Streptomyces peucetius var. caesius)中ε-rhodomycinone产量最高，易于获得^{[14, 24]}。因此早期有较多实验直接以ε-rhodomycinone为底物进行化学修饰或组合生物合成以期获得更有价值的衍生物^{[22, 25]}。关于ε-rhodomycinone天然的非糖基化衍生物报道主要在2010年之前。在1991年，Johdo等^[26-27]从链霉菌(Streptomyces violaceus) A262中分离得到ε-rhodomycinone (1)以及4个类似物β-isorhodomycinone (3)、γ-isorhodomycinone (4)、α-citromycinone (5)、α₂-rhodomycinone (6) (图1)。

本文从中药植物青风藤分离得到一株可产ε-rhodomycinone及其类似物的内生链霉菌SZC001，通过全基因组测序及antiSMASH分析发现其有较大挖掘潜力。本文通过结构鉴定以及活性测试，对其中的蒽环类天然产物进行了初探。

1 材料与方法

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1.1 培养基

SZC001的分离培养基为ISP4培养基和G1培养基^[28]；种子培养基为胰蛋白胨大豆肉汤TSB培养基^[28]；发酵培养基为LNM培养基^[29]；其他测试培养基为T₁培养基^[30]、ME培养基^[31]和Fish培养基^[28]。

1.2 主要试剂和仪器

分析纯石油醚、乙酸乙酯等有机试剂，成都市科隆化学品有限公司；色谱纯乙腈，北京迈瑞达科技有限公司；柔红霉素，上海迈瑞尔生化科技有限公司；DA201树脂，天津浩聚树脂科技有限公司；100−200目硅胶，青岛鼎康硅胶有限公司；Sephadex LH-20凝胶，青岛海洋化工有限公司；分析型色谱柱(250 mm×4.6 mm，5 µm)，沃特世科技(上海)有限公司；半制备色谱柱(250 mm×10 mm，5 µm)，月旭科技(上海)股份有限公司；细菌基因组DNA提取试剂盒，生工生物工程(上海)股份有限公司；PCR试剂、引物合成和测序由北京擎科生物技术有限公司完成。

400 MHz核磁共振波谱仪，布鲁克(北京)科技有限公司；高分辨质谱仪、紫外可见分光光度计、红外光谱仪，赛默飞世尔科技(中国)有限公司；高效液相色谱仪、半制备液相色谱仪，沃特世科技(上海)有限公司；旋转蒸发仪，东京理化器械株式会社；生化培养箱，上海博迅实业有限公司；振荡培养箱，上海知楚仪器有限公司；PCR仪，艾本德股份公司。

1.3 菌株分离

SZC001菌株经表面消毒法分离于野生青风藤须根部(采样地位于湖南省怀化市中方县黄岩镇，地理位置为27°28′14.5′′N，110°3′3.88′′E)。取青风藤置于培养箱100 ℃干燥30 min，去除表面微生物。将其根茎叶拆分，根部的处理：取5%次氯酸钠溶液及10%硫代硫酸钠依次浸泡10 min和5 min。最后用10%碳酸氢钠溶液浸泡5 min，再用无菌水洗净。将须根剪成小段碎末状，均匀散布于5种放线菌培养基(25 mg/L萘啶酮酸及50 mg/L制霉菌素)，置于培养箱28 ℃培养3−7 d；用接种针将单个菌落接种于对应平板进行反复划线直至纯化^[31]。SZC001保藏于中药合成生物学研究湖南省重点实验室内生菌菌株保藏库。

1.4 菌株鉴定及全基因组测序

使用试剂盒提取菌株总DNA，并用引物16S rRNA-F1 (5′-CAGAGTTTGATCCTGGCT-3′)和16S rRNA-R1 (5′-AGGAGGTGATCCAGCCG CA-3′)进行PCR扩增，将对应片段进行16S rRNA基因测序^[31]。测序结果参考美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)数据库进行种属鉴定。菌株SZC001的全基因组测序由北京百迈客生物科技有限公司完成，包括样品质量检测、文库构建、文库质量检测、文库测序以及信息分析等流程。通过Nanopore三代测序平台进行全基因组测序并进行contigs拼接；利用Canu v1.5软件对过滤后的paired-end reads进行组装；通过Racon v3.4.3的三代reads进行结果矫正；通过Circlator v1.5.5进行环化并调整起始位点，最后利用Pilon v1.22软件及二代测序数据进行矫正纠错^[32-33]。使用antiSMASH v7.0进行次级代谢产物基因簇分析^[34]。采用eggNOG、KEGG、Pfam、Swiss-Prot数据库比对测序结果并进行基因功能注释。

1.5 发酵及分离鉴定

取SZC001冻存孢子接种到TSB培养基，28 ℃、220 r/min培养24−48 h。然后按发酵培养基体积分数10%的量转接到LNM DA201液体培养基中，28 ℃、220 r/min培养7 d，共收集发酵液31.5 L。树脂用乙酸乙酯提取，将乙酸乙酯部分真空浓缩得到粗品45.02 g。粗浸膏采用硅胶层析柱(100−200目)进行粗分离，石油醚-乙酸乙酯(1:0→0:1)梯度洗脱，经HPLC分析合并得8个组分Fr.1−Fr.8。通过硅胶柱色谱对组分Fr.5 (1.51 g)进行分离，石油醚-乙酸乙酯(1:0→0:1)洗脱，分离得到了Fr.5.2。Fr.5.2通过Sephadex LH-20凝胶柱色谱，甲醇-二氯甲烷(1:1)洗脱，得到Fr.5.2.2。Fr.5.2.2用半制备型HPLC制备(乙腈-1%甲酸水40:60)得到化合物1 (t_R=12.7 min，10.1 mg)和化合物2 (t_R=11.4 min，12.7 mg)。

1.6 CCK-8 (cell counting kit-8)法测试细胞毒性

使用CCK-8 (上海碧云天生物技术股份有限公司)法对1和2进行细胞毒性测试^[35]。测试细胞系为人胃腺癌细胞SGC-7901和人乳腺癌细胞MDA-MB-231，由上海交通大学张建明课题组提供。96孔板每孔加入100 μL测试细胞，使细胞数约为5 000个。细胞置于37 ℃、5% CO₂细胞培养箱中培养24 h，使细胞贴壁。每孔分别加入32、16、8、4、2、1、0.5、0.125 μg/mL的化合物，置于细胞培养箱中孵育。MDA-MB-231和SGC-7901分别孵育72 h和96 h。酶标仪测定450 nm处的吸光度，按公式(1)计算细胞存活率。

(1)

使用GraphPad Prism 8计算IC₅₀。二甲亚砜(dimethyl sulfoxide, DMSO)作为阴性对照，空培养基作空白对照，柔红霉素作为阳性对照。

2 结果与分析

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2.1 分离鉴定SZC001及全基因组测序分析

本研究利用表面消毒法从青风藤须根中分离得到内生菌SZC001 (原始编号C43-3-3)以及5种其他形态的内生菌(附图1，数据已提交国家微生物科学数据中心，编号：NMDCX0001715)。在G1培养基30 ℃培养，菌体正面从粉红色逐渐变为紫红色，约第3天产生白色孢子，第7天时可完全覆盖菌体表面；菌体背面为粉红色且与培养基紧密结合(图2)。抗生素测试显示它对安普霉素(50 µg/mL)、卡那霉素(50 µg/mL)、硫链丝菌素(50 µg/mL)、利福霉素(50 µg/mL)、庆大霉素(50 µg/mL)均敏感(附图2，数据已提交国家微生物科学数据中心，编号：NMDCX0001715)。以其基因组为模板，通过16S rRNA基因的PCR扩增及分子测序得到一条1 496 bp的基因序列(NCBI登录号为PP702368)。NCBI数据库BLAST分析表明，SZC001与链霉菌(Streptomyces violarus) strain MMS21-305、链霉菌(Streptomyces violarus) strain ISP 5205、链霉菌(Streptomyces violarus) strain NBRC 13104相似度分别为99.72%、99.59%、99.32%，因此将其初步鉴定为链霉菌(Streptomyces sp.) SZC001。对该菌株进行全基因组测序结果显示，read条数120 644，总碱基数1 224 905 477，平均序列读长10 153 bp；测序序列N50和N90的长度分别为21 200 bp和3 731 bp；重复序列为155 777 bp，占总长的1.71%。进一步分析后可知其基因组总长度为9 109 166 bp，G+C含量为71.08% (NCBI登录号为SRR29081433)。生物信息学分析显示基因组含有8 044个编码基因，分别在eggNOG、KEGG、Pfam、Swiss-Prot数据库提取到6 223、2 886、6 636、4 036个基因的注释信息。

2.2 基因簇分析及生物合成潜力预测

基于antiSMASH 7.0对其代谢产物进行分析，结果显示该菌株共含有31个潜在基因簇，包括非核糖体多肽合成酶(non-ribosomal peptide synthetase, NRPS)类基因簇、聚酮合成酶(polyketide synthases, PKS)类基因簇、萜烯(terpene)类化合物合成基因簇、核糖体合成和翻译后修饰多肽(ribosomally synthesized and post-translationally modified peptides, RiPP)类基因簇等类别(表1)。其中9个基因簇与alkylresorcinol、ε-poly-L-lysine、ectoine等已知基因簇的相似度为100%；3个基因簇与已知基因簇相似度为80%−95%；2个基因簇与已知的基因簇相似度为55%−60%；另外有17个生物合成基因簇与已知基因簇的相似度小于40%。其中相似度在30%−40%的基因簇有2个，相似度在10%−25%的基因簇有5个，相似度在10%以下的基因簇有10个，且其中4个基因簇与数据库比对无相似度。表明菌株SZC001具有产生结构新颖或功能独特化合物的潜力。

2.3 化合物分离鉴定及基因簇分析

SZC001在LNM、ME、Fish、T₁四种培养基(添加DA201树脂)中代谢谱较为丰富，主产物均为紫外吸收230、255、290、490 nm的类似物(图3)。其中SZC001在LNM中发酵，该产物产量最高，被用作后期的发酵培养基。

选取LNM进行31.5 L发酵，共得到了45.02 g发酵浸膏。经过硅胶柱层析、凝胶柱、半制备高效液相色谱仪分离纯化共得到发酵含量较高的2个化合物1 (10.1 mg)和2 (12.7 mg)。化合物1为红色粉末状，易溶于二甲亚砜(DMSO)。对该化合物进行液相质谱(LC-MS)分析显示，负离子模式下[M-H]⁻峰为427.102 0，理论值为427.102 3，确定其分子式为C₂₂H₂₀O₉。在氘代DMSO中进行核磁测试。¹H-NMR (400 MHz，DMSO-d₆)波谱数据δ：7.79 (d，J=9.4 Hz，1H，H-1)，7.75 (d，J=7.0 Hz，1H，H-2)，7.37 (d，J=7.7 Hz，1H，H-3)，5.55 (m，1H，H-7)，4.15 (s，1H，H-10)，3.65 (s，3H，H-16)，2.06 (m，2H，H-8)，1.70 (m，1H，H-13α)，1.45 (m，1H，H-13β)，1.05 (t，J=7.3 Hz，3H，H-14)；¹³C-NMR (175 MHz，DMSO-d₆)数据δ：189.77 (C-5)，185.87 (C-12)，171.20 (C-15)，162.16 (C-4)，157.80 (C-11)，156.87 (C-6)，139.90 (C-2)，137.51 (C-10a)，134.35 (C-6a)，133.53 (C-12a)，124.91 (C-3)，119.34 (C-1)，116.82 (C-4a)，111.52 (C-11a)，111.22 (C-5a)，71.51 (C-9)，61.39 (C-7)，52.71 (C-16)，51.82 (C-10)，35.22 (C-13)，32.71 (C-8)，7.29 (C-14)。核磁数据与已报道文献一致，因此化合物1确定为ε-rhodomycinone^[26-27]。

化合物2为橙色粉末状，易溶于二甲亚砜(DMSO)。UV-Vis (MeOH) λmax [lg ε/(L/(mol·cm))]：234 (4.53)、258 (4.28)、294 (3.85)、492 (4.04) nm。该化合物的红外光谱提示结构中存在羟基(3 417.85 cm⁻¹)、共轭羰基(1 719.71 cm⁻¹)和苯环(1 618.74，1 500.34 cm⁻¹)。对其进行液相质谱(LC-MS)分析显示，负离子模式下[M-H]⁻峰为427.101 8，理论值为427.102 3，确定其分子式为C₂₂H₂₀O₉。化合物2的¹H-NMR (400 MHz，DMSO-d₆)波谱数据δ：7.93 (s，1H，H-6)，7.39 (m，1H，H-2)，7.39 (m，1H，H-3)，4.84 (t，J=4.5 Hz，1H，H-7)，4.12 (s，1H，H-10)，3.64 (s，3H，H-16)，2.18 (m，1H，H-8α)，1.98 (m，1H，H-8β)，1.62 (m，1H，H-13α)，1.48 (m，1H，H-13β)，1.03 (t，J=7.2 Hz，3H，H-14)；¹³C NMR (101 MHz，DMSO-d₆)数据δ：189.53 (C-12)，186.35 (C-5)，171.72 (C-15)，161.36 (C-11)，157.88 (C-4)，157.84 (C-1)，149.23 (C-6a)，131.64 (C-5a)，130.73 (C-2)，130.04 (C-10a)，129.81 (C-3)，119.69 (C-6)，114.42 (C-11a)，113.27 (C-4a)，113.11 (C-12a)，71.46 (C-9)，65.95 (C-7)，52.44 (C-16)，51.84 (C-10)，37.89 (C-8)，32.20 (C-13)，7.45 (C-14)。其与化合物1 ¹³C-NMR谱有较高的相似度(表2)，但具有差异，推测化合物2为其类似物。经过归属发现化合物2与α₂-rhodomycinone ¹³C-NMR谱高度相似，但含有特有的171.20 (C-15)及52.71 (C-16)信号^[27]。该信号与化合物1的COOCH₃的C-15及C-16信号相似。比对¹H-NMR谱图也发现，化合物2相对于α₂-rhodomycinone有一个明显的3.64 (3H，s，COOCH₃)信号，因此推测化合物2是α₂-rhodomycinone的C-10位OH被替换为了COOCH₃。据HMBC两组信号4.12 (H-10)→161.36 (C-11)以及7.93 (H-6)→ 65.95 (C-7)，可判定B环OH应该在C-11而非C-6位。通过HMBC及HSQC谱得到进一步确认(图3)。由此确定化合物2为新的化合物结构，命名为η-rhodomycinone。结构解析及文献调研初步判断基因簇14可负责rhodomycinone及其类似物的生物合成，它与已报道的cytorhodin的生物合成基因簇相似度为84%^{[23, 36]}。

2.4 细胞毒性测试

按CCK-8法对化合物1和2进行细胞毒性测试。化合物1对人胃腺癌细胞SGC-7901和人乳腺癌细胞MDA-MB-231具有较好活性，IC₅₀值分别为1.55 μmol/L和3.23 μmol/L。化合物2对人胃腺癌细胞SGC-7901的活性略低于化合物1，IC₅₀值为4.59 μmol/L。其对人乳腺癌细胞MDA-MB-231的抑制与化合物1类似，IC₅₀值为2.11 μmol/L。阳性对照为柔红霉素，它对以上2种肿瘤细胞的IC₅₀均小于0.125 μmol/L。

3 讨论与结论

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基于“内共生”原理，部分内生菌株可产生与宿主相同或相似的代谢产物，代表案例有紫杉醇、鬼臼毒素、喜树碱等，这引发了植物内生菌的研究热潮^[37-38]。有学者指出植物内生菌研究的真正意义不仅在于其生态的独特性，更在于微生物在宿主体内和它们的协同作用及协同作用所产生的新物质和新功能^[2]。越来越多的证据也表明植物内生菌是一类开发不足的微生物资源，从中可发掘具有新颖结构或作用靶点的抗生素^[5-8]。

本研究初步判定基因簇14负责编码化合物1和2及其类似物的生物合成。尽管该基因簇与已知基因簇cytorhodin相似度较高(84%)，但SZC001基因组中还有其他相似度较低的次级代谢产物可被挖掘。如friulimicin是一种与daptomycin类似但作用靶点不同的脂肽，它们是一类新型抗生素^[39]。Rifamorpholines A−E是最近鉴定的利福霉素的一个新亚类，具有特殊的5/6/6/6环发色团，能有效抗耐甲氧西林金黄色葡萄球菌^[40]。Colabomycin E可显著抑制THP-1细胞释放IL-1β，是一种潜在的抗炎化合物^[41]。

蒽环类天然产物通常可作为嵌入剂及拓扑异构酶抑制剂而发挥抗肿瘤作用^[42-43]。然而，由于剂量依赖性心脏毒性等问题限制了其应用。蒽环类药物结构差异会导致不同的心脏毒性，如柔红霉素较阿霉素的心脏毒性更低，因此有研究者关注它们衍生物改造及活性评估^[17]。从自然界中寻找天然存在的活性更强，毒性更低的衍生物也是一种重要思路。本文对含量最高的产物1和2仅进行了初步的细胞毒性测试，后期可对其他含量较低的类似物进行结构解析并对它们的抗肿瘤活性及细胞毒性进行综合评价。

综上所述，本研究从青风藤的根系中分离得到一株内生菌SZC001，通过全基因组测序评估了其生物合成潜力并对其蒽环类代谢产物进行了初探。化合物的分离鉴定丰富了rhodomycinone的天然产物库，而SZC001则为青风藤内生放线菌的生物活性代谢产物后续发掘提供了基础。

基金

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湖南省自然科学基金(2023JJ50439)
湖南省教育厅科学研究项目(22C1186)
湖南省大学生创新创业训练计划(S2022122140016)

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2024年第64卷第12期

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doi: 10.13343/j.cnki.wsxb.20240370

接收时间：2024-06-17
首发时间：2026-03-21
出版时间：2024-09-27

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收稿日期：2024-06-17
录用日期：2024-09-25

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Natural Science Foundation of Hunan Province(2023JJ50439)

湖南省自然科学基金(2023JJ50439)

Scientific Research Fund of Hunan Provincial Education Department(22C1186)

湖南省教育厅科学研究项目(22C1186)

Hunan Provincial College Students' Innovation and Entrepreneurship Training Program(S2022122140016)

湖南省大学生创新创业训练计划(S2022122140016)

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湖南医药学院, 中药合成生物学研究湖南省重点实验室, 湖南怀化 418000

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Cluster	Position of gene cluster (start)	Position of gene cluster (termination)	Type of gene cluster	Most similar known cluster	Similarity (%)
−: No similar gene cluster predicted.
1	6 891	68 544	T3PKS, NRPS, T1PKS	Alkylresorcinol	100
2	229 260	250 820	Melanin, terpene	Melanin	57
3	643 925	689 547	NRPS-like, T1PKS	Olimycin A/B	20
4	1 188 455	1 208 783	Terpene	Clipibycyclene	6
5	1 362 768	1 402 798	T3PKS	Flaviolin/1,3,6,8-tetrahydro xynaphthalene	100
6	1 921 470	1 953 655	NAPAA	ε-poly-L-lysine	100
7	2 176 155	2 186 553	Ectoine	Ectoine	100
8	2 704 623	2 753 867	T1PKS, NRPS	Rifamorpholine A/B/C/D/E	3
9	2 968 910	2 989 421	Terpene	SCO-2138	14
10	3 256 213	3 291 146	NAPAA	Stenothricin	13
11	3 352 468	3 363 133	Melanin	Melanin	40
12	3 457 262	3 468 035	NI-siderophore	Desferrioxamin B/E	83
13	4 717 147	4 728 088	Butyrolactone	Coelimycin P1	8
14	5 185 780	5 258 286	T2PKS, Oligosaccharide, PKS-like	Cytorhodin	84
15	5 792 861	5 803 409	Butyrolactone	−	−
16	5 813 805	5 824 746	Butyrolactone	Neocarzinostatin	4
17	5 900 336	5 941 140	2dos	Notonesomycin A	6
18	6 189 388	6 209 988	Terpene	Albaflavenone	100
19	6 843 605	6 854 598	NI-siderophore	−	−
20	7 142 887	7 153 846	RiPP-like	−	−
21	7 217 971	7 238 601	Terpene	Geosmin	100
22	7 388 394	7 411 573	RRE-containing	−	−
23	7 481 332	7 619 277	NI-siderophore, NRPS, NRPS-like	Friulimicin A/B/C/D	33
24	8 025 082	8 052 261	Terpene	Hopene	92
25	8 199 733	8 258 068	NRP-metallophore, NRPS	Coelichelin	100
26	8 393 130	8 434 467	Ladderane	Colabomycin	15
27	8 465 947	8 507 131	T3PKS	Germicidin	100
28	8 572 827	8 586 380	NI-siderophore	Peucechelin	25
29	8 711 211	8 738 249	RiPP-like, lanthipeptide-class-iii	Informatipeptin	100
30	8 800 286	8 844 260	T1PKS	Chlorothricin/Deschlorothricin	4
31	8 914 379	8 958 647	NRPS	Diisonitrile antibiotic SF2768	55

Cluster

Position of gene cluster (start)

Position of gene cluster (termination)

Type of gene cluster

Most similar known cluster

Similarity (%)

−: No similar gene cluster predicted.

6 891

68 544

T3PKS, NRPS, T1PKS

Alkylresorcinol

100

229 260

250 820

Melanin, terpene

Melanin

643 925

689 547

NRPS-like, T1PKS

Olimycin A/B

1 188 455

1 208 783

Terpene

Clipibycyclene

1 362 768

1 402 798

T3PKS

Flaviolin/1,3,6,8-tetrahydro xynaphthalene

100

1 921 470

1 953 655

NAPAA

ε-poly-L-lysine

100

2 176 155

2 186 553

Ectoine

100

2 704 623

2 753 867

T1PKS, NRPS

Rifamorpholine A/B/C/D/E

2 968 910

2 989 421

Terpene

SCO-2138

3 256 213

3 291 146

NAPAA

Stenothricin

3 352 468

3 363 133

Melanin

3 457 262

3 468 035

NI-siderophore

Desferrioxamin B/E

4 717 147

4 728 088

Butyrolactone

Coelimycin P1

5 185 780

5 258 286

T2PKS, Oligosaccharide, PKS-like

Cytorhodin

5 792 861

5 803 409

Butyrolactone

−

5 813 805

5 824 746

Butyrolactone

Neocarzinostatin

5 900 336

5 941 140

2dos

Notonesomycin A

6 189 388

6 209 988

Terpene

Albaflavenone

100

6 843 605

6 854 598

NI-siderophore

−

7 142 887

7 153 846

RiPP-like

−

7 217 971

7 238 601

Terpene

Geosmin

100

7 388 394

7 411 573

RRE-containing

−

7 481 332

7 619 277

NI-siderophore, NRPS, NRPS-like

Friulimicin A/B/C/D

8 025 082

8 052 261

Terpene

Hopene

8 199 733

8 258 068

NRP-metallophore, NRPS

Coelichelin

100

8 393 130

8 434 467

Ladderane

Colabomycin

8 465 947

8 507 131

T3PKS

Germicidin

100

8 572 827

8 586 380

NI-siderophore

Peucechelin

8 711 211

8 738 249

RiPP-like, lanthipeptide-class-iii

Informatipeptin

100

8 800 286

8 844 260

T1PKS

Chlorothricin/Deschlorothricin

8 914 379

8 958 647

NRPS

Diisonitrile antibiotic SF2768

Compound 1 (ε-rhodomycinone)		Compound 2 (η-rhodomycinone)
1	119.34, CH	7.79, d (9.4)	157.84, C
2	139.90, CH	7.75, d (7.0)	130.73, CH	7.39, m	4
3	124.91, CH	7.37, d (7.7)	129.81, CH	7.39, m
4	162.16, C		157.88, C
4a	116.82, C		113.27, C
5	189.77, C		186.35, C
5a	111.22, C		131.64, C
6	156.87, C		119.69, CH	7.93, s	7, 11a, 10a, 5
6a	134.35, C		149.23, C
7	61.39, CH	5.55, m	65.95, CH	4.84, t (4.5)
8	32.71, CH₂	2.06, m	37.89, CH₂	Hα 2.18, m	6a, 7, 9
				Hβ 1.98, m
9	71.51, C		71.46, C
10	51.82, CH	4.15, s	51.84, CH	4.12, s	8, 9, 10a, 6a, 11, 15
10a	137.51, C		130.04, C
11	157.80, C		161.36, C
11a	111.52, C		114.42, C
12	185.87, C		189.53, C
12a	133.53, C		113.11, C
13	35.22, CH₂	Hα 1.70, m	32.20 CH₂	Hα 1.62, m	14, 8, 10, 9
		Hβ 1.45, m		Hβ 1.48, m
14	7.29, CH₃	1.05, t (7.3)	7.45, CH₃	1.03, t (7.2)	13, 9
15	171.20, C		171.72, C
16	52.71, CH₃	3.65, s	52.44, CH₃	3.64, s	15

Compound 1 (ε-rhodomycinone)

Compound 2 (η-rhodomycinone)

Position

δ_C, type

δ_H, multiplicity (J in Hz)

δ_C, type

δ_H, multiplicity (J in Hz)

HMBC

119.34, CH

7.79, d (9.4)

157.84, C

139.90, CH

7.75, d (7.0)

130.73, CH

7.39, m

124.91, CH

7.37, d (7.7)

129.81, CH

7.39, m

162.16, C

157.88, C

116.82, C

113.27, C

189.77, C

186.35, C

111.22, C

131.64, C

156.87, C

119.69, CH

7.93, s

7, 11a, 10a, 5

134.35, C

149.23, C

61.39, CH

5.55, m

65.95, CH

4.84, t (4.5)

32.71, CH₂

2.06, m

37.89, CH₂

Hα 2.18, m

6a, 7, 9

Hβ 1.98, m

71.51, C

71.46, C

51.82, CH

4.15, s

51.84, CH

4.12, s

8, 9, 10a, 6a, 11, 15

10a

137.51, C

130.04, C

157.80, C

161.36, C

11a

111.52, C

114.42, C

185.87, C

189.53, C

12a

133.53, C

113.11, C

35.22, CH₂

Hα 1.70, m

32.20 CH₂

Hα 1.62, m

14, 8, 10, 9

Hβ 1.45, m

Hβ 1.48, m

7.29, CH₃

1.05, t (7.3)

7.45, CH₃

1.03, t (7.2)

13, 9

171.20, C

171.72, C

52.71, CH₃

3.65, s

52.44, CH₃

3.64, s