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Journal of Tropical Oceanography >

2023 , Vol. 42 >Issue 3: 169 - 173

DOI: https://doi.org/10.11978/2022107

Marine Geology

The isolation and identification of neoabyssomicin H from the deep-sea derived Streptomyces koyangensis SCSIO 5802*

  • SONG Yongxiang , 1, 2, 3, 4 ,
  • LI Xiaoyue 1, 3 ,
  • JU Jianhua 1, 2, 3, 4
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  • 1. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
  • 2. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511453, China
  • 3. University of Chinese Academy of Sciences, Beijing 100049, China
  • 4. Sanya Institute of Oceanology, SCSIO, Sanya 572000, China
SONG Yongxiang. email:

Copy editor: LIN Qiang

Received date: 2022-05-12

  Revised date: 2022-06-13

  Online published: 2022-06-16

Supported by

Finance Science and Technology Project of Hainan Province(ZDKJ202018)

Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou)(GML2019ZD0406)

Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(2019BT02Y262)

Guangdong Provincial Marine Economic Development (Six Major Marine Undertakings) Special Fund Project(GDNRC[2021]54)

National Natural Science Foundation of China(41676151)

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Abstract

Abyssomicins have attracted much attention for their potential of being developed into novel lead compounds by virtue of their antimicrobial and antiviral activities. The deep-sea derived strain of Streptomyces koyangensis SCSIO 5802 produces a series of abyssomicins. In order to further expand the library of abyssomicins, the fermentation fractions of this strain have been thoroughly investigated, which led to the first isolation of a new thioether dimer of abyssomicin with a macrolide moiety named neoabyssomicin H. Its structure was elucidated by using UV, IR, HR-ESI-MS, 1D and 2D NMR, and X-ray single crystal using Cu Kα radiation, etc. In antibacterial activity assays, the compound was found inactive against Staphylococcus aureus ATCC 29213 and a panel of MRSA clinical isolates at 128 μg·mL-1. This study has expanded the scope of abyssomicins with a new molecular entity for study on this class of lead compounds.

Key words： deep-sea derived actinomycete; abyssomicin; antibacteria; thioether dimerization

Cite this article

SONG Yongxiang , LI Xiaoyue , JU Jianhua . The isolation and identification of neoabyssomicin H from the deep-sea derived Streptomyces koyangensis SCSIO 5802*[J]. Journal of Tropical Oceanography, 2023 , 42(3) : 169 -173 . DOI: 10.11978/2022107

深渊霉素类(abyssomicins)化合物, 最早分离自289m深的一株海底沉积物放线菌Verrucosispora strain AB 18-032 (Riedlinger et al, 2004)。该类化合物为多环聚酮结构, 对革兰氏阳性细菌包括结核分枝杆菌(Freundlich et al, 2010)、金黄色葡萄球菌及其耐药菌MRSA(methicillin-resistant Staphylococcus aureus) (Bister et al, 2004)等具有抑制活性, 对潜伏的HIV病毒具有激活作用(León et al, 2015)。深渊霉素类化合物的抗菌机理为抑制对氨基苯甲酸/四氢叶酸的生物合成, 该生物合成途径是许多微生物和植物的专有途径, 因在人体中不存在, 被认为是理想靶标(Bister et al, 2004; Riedlinger et al, 2004)。目前已发现的近40个深渊霉素类化合物, 根据其结构的对应关系主要分为Ⅰ型和Ⅱ型两大类型, 其中Ⅱ型的数量相对较多(Song et al, 2017)。
前期我们对一株来自3536m深的海底沉积物放线菌Streptomyces koyangensis SCSIO 5802的次级代谢产物进行研究, 先后发现一系列结构新颖的深渊霉素类化合物(1~11, 图1), 包括新骨架化合物6/6/6环骈6/9内酯环结构的neoabyssomicin A(Song et al, 2017)、8/5/5/7多环结构的neoabyssomicin D及两个硫醚二聚体化合物neoabyssomicin F和G(Huang et al, 2018)。随后, 该类化合物的生物合成基因簇和生物合成机制也得到揭示(Li et al, 2018; Tu et al, 2018; Ji et al, 2020)。为进一步加强对深渊霉素类化合物的发掘, 本文通过对深渊霉素生产菌株SCSIO 5802的发酵组分进行检测、分离和结构鉴定, 发现了一个结构新颖的大环内酯类深渊霉素硫醚二聚体化合物neoabyssomicin H, 并测试了该化合物对金黄色葡萄球菌和系列MRSA致病细菌的抑制作用。
图1 来自南海深海放线菌的深渊霉素1~11(来自Streptomyces koyangensis SCSIO 5802)和abyssomicin J (来自Verrucosispora sp. MS100128)

Fig. 1 The abyssomicins 1~11

1 材料与方法

1.1 仪器与试剂

ACB-4A1超净工作台(ESCO); 旻泉MQD-B2NR 组合式振荡培养箱(上海旻泉); Centrifuge 5810 R离心机(Eppendorf); Laborate 4000 efficient旋转蒸发仪(Heidolph); Agilent 1260高效液相色谱分析仪(Agilent Technologies); 半制备高效液相色谱(Hitachi, HPLC); 超低温超导核磁共振波谱仪(Bruker AVANCE Ⅲ-700 MHz); 超高分辨液-质联用仪(Bruker maXis QTOF); 旋光仪(Anton Paar MCP-500); 紫外可见分光光度计(UV-2600); 傅里叶红外光谱仪(IR Affinity-1); 微焦斑铜转靶的单晶衍射仪(XtaLAB PRO MM007HF X-ray)。
色谱纯乙腈(百灵威科技); 冰醋酸(分析纯、天津市大茂化学试剂厂); 甲醇、氯仿、丙酮、丁酮、乙酸乙酯、石油醚(分析纯、广州化学试剂厂); 超纯水等。

1.2 研究菌株及其放大发酵

深海放线菌Streptomyces koyangensis SCSIO 5802分离自3536 m深的海底沉积物, 保存于中国科学院南海海洋研究所。
从-80℃冰箱中取出菌种, 解冻后三线法划线接种于MISP2平板(葡萄糖0.4%, 酵母提取粉0.4%, 麦芽提取粉1%, 海盐3%, pH 7.2~7.4, 琼脂粉1.8%), 置于28℃培养箱培养5~7d, 以活化菌种。将活化的菌种转接于含有50mL RA培养基 (葡萄糖1%, 麦芽提取粉1%, 玉米粉0.5%, 可溶性淀粉2%, 麦芽糖1%, 微量元素100μL·L-1, 海盐3%, pH 7.2~7.4, CaCO3 0.2%) 的250mL摇瓶中, 在28 ℃, 200r·min-1下培养36 h, 获得种子液。每瓶种子液分别转接于含有200mL RA培养基的1 L摇瓶中, 合计18 L发酵液于28 ℃, 200r·min-1发酵10 d后收菌。

1.3 目标化合物的提取与分离

发酵产物以3600r·min-1离心10min, 得发酵液上清和菌体沉淀。对发酵液上清, 使用等体积的丁酮萃取三次; 对发酵产生的菌体, 使用没过菌体的丙酮超声萃取三次, 分别浓缩获得菌体(M)、菌液(S)粗提物浸膏。对M和S浸膏分别以氯仿/甲醇(100/0, 98/2, 96/4, 94/6, 92/8, 90/10, 80/20, 50/50)进行正相硅胶分离, 得MA1~MA8和SA1~SA8各8个组分。分别进行HPLC-DAD(high performance liquid chromatography-diode array detection)全波长指纹图谱检测后, 合并SA1和SA2进一步以石油醚/乙酸乙酯(100/0, 90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 30/70)进行硅胶色谱分离纯化, 得B1~B8共8个组分。HPLC-DAD指纹图谱检测显示, 在B6组分中有一个紫外吸收与深渊霉素相似的未知小峰, 对该峰进行(+)-HR-ESI-MS检测显示, 该化合物的[M+Na]+离子峰为m/z 781.2535, 分子式为C38H46O14SNa, 为未发现的分子, 推测很可能是一个硫醚二聚体类化合物。对该组分以水/乙腈进行SP-HPLC(semi-preparative high performance liquid chromatography)半制备(色谱柱: YMC ODS-A, 250 × 10mm, 5μm; 分离条件: 80/20~0/100, 0~17min, 2.5mL·min-1, 270nm), 在tR = 15.1min时得到目标化合物约1.2mg。

1.4 抑菌活性测试

抑菌实验的MIC(minimum inhibitory concentration)值测定采用CLSI标准的二倍稀释法(CLSI, 2012)。最大起始浓度为128μg·mL-1。活性测试细菌采用金黄色葡萄球菌Staphylococcus aureus ATCC 29213及金黄色葡萄球菌的系列临床耐甲氧西林菌株MRSA-669、1682、3096和A1。

2 结构鉴定与抗菌活性

2.1 硫醚二聚体化合物neoabyssomicin H的结构鉴定

Neoabyssomicin H, 无色方晶, 非晶体状态时为白色粉末。根据(+)-HR-ESI-MS加钠分子离子峰m/z 781.2535确定其分子式为C38H46O14S。而其NMR数据显示, 该化合物仅有19个碳信号(表1), 因此推测该化合物为一个硫醚二聚体。通过分析对比其NMR数据发现, 该化合物与之前从该菌中发现的新深渊霉素neoabyssomicin B (2)非常相似(Song et al, 2017), 但该化合物中没有化合物2在C-8、C-9位的不饱和双键, 而在δC 37.2和δC 38.8处分别多出一个亚甲基和一个次甲基信号, 推断该化合物结构为neoabyssomicin B (2)在C-8、C-9位处不饱和双键被还原, 并在C-9位通过硫原子连接而成的二聚体化合物。进而, 通过HMBC的H3-17/17'到C-5/5', C-6/6'; H-6/6'到C-7/7'; H-4/4'到C-3/3', C-6/6'等的相关, 确定了其大环内酯结构的存在。通过H-10/10'到C-9/9', C-11/11', C-15/15', C16/16'; H3-18到C-11/11', C-12/12', C-13/13'; H3-19到C-12/12', C-13/13', C-14/14'等的相关, 证实了其与化合物2中各环结构的一致。尤其是从H-9/9'(δH 2.79)与C-9'/9(δC 38.8)的HMBC相关, 进一步确认了该化合物是通过C-9位以硫醚键连接而成硫醚二聚体结构, 从而解析了该化合物的平面结构(如图2和表1)。根据深渊霉素的生物合成机制, 推测该化合物的绝对构型与化合物2一致。幸运的是, 以丙酮溶解该化合物培养单晶, 获得了较高质量的晶体, 以X-Ray单晶铜靶衍射的方式解析了其绝对构型[Flack 0.030 (14)], 证实与推测结果一致。该化合物为一个首次发现的大环内酯类深渊霉素硫醚二聚体, 命名为新深渊霉素H, 即neoabyssomicin H(图2、3)。
表1 新深渊霉素H在氘代甲醇中的1H(700MHz)、13C NMR(175MHz)和HMBC数据(δ, ppm, J, Hz)

Tab. 1 The 1H (700 MHz), 13C NMR (175 MHz) spectroscopic data and HMBC correlations of neoabyssomicin H in CD3OD (δ in ppm, J in Hz)

原子序号 碳谱化学位移δC 氢谱化学位移δH HMBC相关数据
1/1', C 170.9
2/2', C 100.9
3/3', C 194.4
4/4', CH2 37.4 2.97, ddd (13.1, 6.5, 3.5) 5/5',
2.64, m 6/6', 3/3'
5/5', CH2 32.9 2.08, m 3/3'
2.02, m 4/4', 3/3'
6/6', CH 73.2 4.85, overlapped
7/7', C 170.2
8/8', CH2 37.2 2.81, overlapped 7/7', 9/9', 10/10'
2.66, overlapped 9/9'
9/9', CH 38.8 2.79, overlapped 7/7', 8/8', 9'/9, 15/15'
10/10', CH 51.4 2.68, dd (5.0, 4.3) 9/9', 11/11', 15/15', 16/16'
11/11', CH 71.1 4.51, d (4.3) 10/10', 9/9', 12/12', 18/18', 13/13'
12/12', C 90.6
13/13', CH 27.8 2.62, m 15/15', 19/19'
14/14', CH2 37.1 2.81, overlapped 19/19', 13/13', 10/10', 15/15', 16/16'
2.40, dd (17.5, 5.5) 1/1', 2/2', 13/13'
15/15', C 77.0
16/16', C 186.6
17/17', CH3 18.9 1.22, d (6.2) 5/5', 6/6'
18/18', CH3 18.1 1.57, s 11/11', 12/12', 13/13'
19/19', CH3 15.5 1.01, d (7.1) 12/12', 13/13', 14/14'
图2 新深渊霉素H及其关键的HMBC相关

Fig. 2 Structure of neoabyssomicin H and its key HMBC correlations

图3 新深渊霉素H的晶体结构

Fig. 3 X-ray crystallographic structure of neoabyssomicin H

新深渊霉素H, [ α ] D 25 = + 11.0 (c 0.12, MeOH); UV (MeOH): λmax (log ε) = 256 (3.69), 206 (3.62) nm; IR : ṽmax=3371, 1633cm-1. 1H NMR(700 MHz)和13C NMR(175 MHz)数据如表1。(+)-HR-ESI-MS m/z [M+NH4]+ 776.2955 (Calcd C38H50NO14S, 776.2947), [M+Na]+ 781.2535 (Calcd C38H46NaO14S, 781.2500).
新深渊霉素H的晶体数据: 单斜晶系, P21 空间群, a = 7.5971(2) Å, b = 13.8843(4) Å, c = 18.5887(5) Å, α = 90, β = 90.184(3)°, γ = 90, V = 1960.73(9) Å3, Z = 2, T = 100.00 (10)K, μ(Cu Kα) = 1.352mm-1, Dcalc = 1.384g·cm-3, 收集总衍射点19714 (6.366°≤2Θ≤ 148.732°), 独立衍射点7733 (Rint = 0.0459, Rsigma = 0.0461). 最终参数R1: 0.0584 [I>2σ(I)] 和 wR2: 0.1624 (所有数据), Flack 参数为0.030(14)。晶体数据保存于剑桥晶体数据中心, 保藏号CCDC 2167714。

2.2 抗菌活性结果

将新深渊霉素H以二倍稀释法, 以万古霉素为对照, 对金黄色葡萄球菌Staphylococcus aureus ATCC 29213及其系列临床耐甲氧西林菌包括MRSA-669, MRSA-1862, MRSA-3096和MRSA-A1的抑制活性, 进行MIC值的测定。结果新深渊霉素H在最大浓度128μg·mL-1时, 对所测菌株均未发现抑制活性(阳性对照万古霉素的MIC值依次为0.5、1、1、0.5和大于128μg·mL-1)。

3 结论与讨论

本文对深海放线菌Streptomyces koyangensis SCSIO 5802产深渊霉素类化合物进行深入发掘, 发现了一个硫醚二聚体类化合物, 该化合物是首次发现的一个以大环内酯深渊霉素类单体形成的硫醚二聚体, 将其命名为新深渊霉素H。通过对新深渊霉素H进行系列抗金黄色葡萄球菌及系列临床MRSA菌株的抑制活性测试, 在最大浓度为128μg·mL-1时, 未发现其对所测试菌株的抑制活性。而深渊霉素硫醚二聚体abyssomicin J(图1)被发现具有前体药物的功能, 其在体外相对稳定和安全, 在体内经过逆迈克尔加成反应, 转化为具有迈克尔受体的单体结构, 可形成活性更高的产物, 以发挥其抗菌效果(Bister et al, 2004)。根据现有构效关系推测, 两个二聚体活性的差异, 可能主要是由于Baeyer Villiger反应在新深渊霉素H的C-6/6'和C-7/7'位处插入的氧原子, 降低了降解后的单体分子在体内的活性(Song et al, 2017)。尽管如此, 新深渊霉素H的发现也为硫醚二聚体类先导化合物的进一步研究提供了新的分子实体。

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