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Article(id=1200394153394819631, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200394147019477416, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2024-0089, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1706284800000, receivedDateStr=2024-01-27, revisedDate=1715011200000, revisedDateStr=2024-05-07, acceptedDate=null, acceptedDateStr=null, onlineDate=1764125868414, onlineDateStr=2025-11-26, pubDate=1720713600000, pubDateStr=2024-07-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764125868414, onlineIssueDateStr=2025-11-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764125868414, creator=13701087609, updateTime=1764125868414, updator=13701087609, issue=Issue{id=1200394147019477416, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='7', pageStart='1897', pageEnd='2182', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764125866894, creator=13701087609, updateTime=1764225115484, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200810425920115296, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200394147019477416, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200810425920115297, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200394147019477416, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2161, endPage=2170, ext={EN=ArticleExt(id=1200394153814250048, articleId=1200394153394819631, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Heterologous expression and product identification of diterpene synthase involved in the biosynthesis of brasilicardin A, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Brasilicardin A, a diterpene glycoside isolated from pathogenic actinomycete Nocardia brasiliensis IFM 0406, has become a novel immunosuppressant candidate due to its significant immunosuppressive activity, low toxicity and unique mechanism of action. However, brasilicardin A and its analogues have become a research hotspot to the development of this promising immunosuppressant because of the low-yield production in the natural pathogenic producer and the synthetically challenging skeleton. According to the reported biosynthetic pathway of brasilicardin A, the function of involved diterpene synthase was analyzed by bioinformatics. Then the genes bra1-5 that synthesize the brasilicardin A skeleton were directionally amplified from the pathogenic strain N. brasiliensis IFM 0406, and heterologous expression was achieved successfully in Streptomyces albus R1. The compounds were isolated and purified by using various column chromatographies including silica gel column chromatography and semi-preparative HPLC. Six new brasilicardins were established and named brasilicardin H-M. The activity of brasilicardins was screened using lipopolysaccharide (LPS)-activated mouse primary macrophage inflammation model. Brasilicardin H-M exhibited good inhibitory activity on nitric oxide (NO) release with IC50 values of 28.24 ± 3.70, 37.44 ± 2.00, 39.85 ± 4.02, 26.77 ± 4.40, 65.25 ± 1.48 and 15.24 ± 2.72 μmol·L-1, respectively (indomethacin as the positive control with IC50 value of 34.28 ± 4.10 μmol·L-1). The results indicated that six compounds had potential anti-inflammatory activity. This study laid a foundation for the elucidation of the brasilicardin A biosynthetic pathway and evaluation of the structure-activity relationship as well as new drug developments.

, correspAuthors=Juan WANG, She-po SHI, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Xiang-yu GE, Guang-xin ZHOU, Na XIONG, Zi-han LU, Xin-yu MI, Zhi-xiang ZHU, Xiao LIU, Xiao-hui WANG, Juan WANG, She-po SHI), CN=ArticleExt(id=1200394157337465556, articleId=1200394153394819631, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=Brasilicardin A生物合成相关二萜合酶的异源表达及产物鉴定, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

从致病性放线菌Nocardia brasiliensis IFM 0406中得到的二萜糖苷brasilicardin A, 因免疫抑制活性显著、毒性较低且作用机制独特而成为新型免疫抑制剂的候选分子。由于原菌株中产率低, 且结构复杂、化学合成困难, brasilicardin A及其类似物的获得成为该类新型免疫抑制剂的研究热点。根据目前报道的brasilicardin A可能的生物合成途径, 通过生物信息学分析生物合成相关二萜合酶, 定向从致病菌株N. brasiliensis IFM 0406中扩增可能合成brasilicardin A骨架的基因bra1~5, 在白色链霉菌Streptomyces albus R1中成功实现异源表达。通过液体发酵培养, 利用硅胶柱色谱及半制备液相色谱等方法进行化合物的分离纯化, 共分离到6个新brasilicardins化合物, 命名为brasilicardin H~M。利用脂多糖(LPS) 活化的小鼠原代巨噬细胞炎症模型对分离鉴定的brasilicardins进行了体外活性筛选, 结果显示, brasilicardin H~M具有较强抑制巨噬细胞释放一氧化氮(NO) 的作用, IC50分别为28.24 ± 3.70、37.44 ± 2.00、39.85 ± 4.02、26.77 ± 4.40、65.25 ± 1.48、15.24 ± 2.72 μmol·L-1 (阳性对照吲哚美辛IC50为34.28 ± 4.10 μmol·L-1), 表明6个化合物具有潜在的抗炎活性。本研究为brasilicardin A生物合成途径的彻底阐明提供了条件, 也为研究该类化合物的构效关系、药物研发等奠定了基础。

, correspAuthors=王娟, 史社坡, authorNote=null, correspAuthorsNote=
*王娟, Tel: 86-10-53911868, E-mail: ;
史社坡, E-mail:
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Appl Microbiol Biotechnol, 2016, 100: 10563-10572., articleTitle=null, refAbstract=null), Reference(id=1200470905563509518, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[21], rfOrder=20, authorNames=null, journalName=null, refType=null, unstructuredReference=Myronovskyi M, Rosenkränzer B, Nadmid S, et al. Generation of a cluster-free Streptomyces albus chassis strains for improved heterologous expression of secondary metabolite clusters [J]. Metab Eng, 2018, 49: 316-324., articleTitle=null, refAbstract=null), Reference(id=1200470905664172816, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[22], rfOrder=21, authorNames=null, journalName=null, refType=null, unstructuredReference=Novakova R, Núñez LE, Homerova D, et al. Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains [J]. 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The plausible biosynthetic pathway of brasilicardin H-M (G) , figureFileSmall=4XyczWdcjIp8h8cYbs8WMA==, figureFileBig=hdJhp1y4NlnsB+hQNjuc+A==, tableContent=null), ArticleFig(id=1200470902073848470, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Category nameDescription
StrainsE. coli DH5aGeneral cloning host
E. coli ET12567 (pUZ8002)Cmr, Kmr, donor strain for conjugation
S. albus R1Wild strain
GXY01The S. albus R1 strain harboring plasmid pGXY1
PlasmidspIB139Integrative plasmid, harboring a PermE* promoter, Aprr, OriTRK2, φC31 int/attP
pGXY1bra1~5 gene under the control of promoter PermE* in plasmid pIB139
Primers (5′-3′)Primer 1taggatccacatatgATGGTGAATTCCGGAGAATGGGTC
Primer 2ccgcggatcctctagaTCAGCACATCGCCTCCTCGA
Primer 3ATGACCACCCGCACGATCGA
Primer 4GCCATCGTCGTCCTTCCTGC
), ArticleFig(id=1200470902191288984, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, language=CN, label=Table 1, caption=

Strains, plasmids and primers used in this study (E. coli: Escherichia coli; S. albus R1: Streptomyces albus R1; Cmr: Chloramphenicol; Kmr: Kanamycin; Aprr: Apramycin sulfate)

, figureFileSmall=null, figureFileBig=null, tableContent=
Category nameDescription
StrainsE. coli DH5aGeneral cloning host
E. coli ET12567 (pUZ8002)Cmr, Kmr, donor strain for conjugation
S. albus R1Wild strain
GXY01The S. albus R1 strain harboring plasmid pGXY1
PlasmidspIB139Integrative plasmid, harboring a PermE* promoter, Aprr, OriTRK2, φC31 int/attP
pGXY1bra1~5 gene under the control of promoter PermE* in plasmid pIB139
Primers (5′-3′)Primer 1taggatccacatatgATGGTGAATTCCGGAGAATGGGTC
Primer 2ccgcggatcctctagaTCAGCACATCGCCTCCTCGA
Primer 3ATGACCACCCGCACGATCGA
Primer 4GCCATCGTCGTCCTTCCTGC
), ArticleFig(id=1200470902329701023, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
No.123
δH mult. (J in Hz)δCδH mult. (J in Hz)δCδH mult. (J in Hz)δC
11.30, m34.6a 1.78, m b 0.96, m29.2a 1.92, m b 1.46, m33.1
21.46, m27.9a 1.80, m b 1.40, m25.9a 2.65, ddd (15.6, 9.0, 5.5) b 2.27, ddd (15.6, 9.0, 5.5)33.7
32.97, m77.13.19, m73.7218.0
439.337.946.5
51.41, m43.01.92, m36.11.90, m47.8
6a 1.58, m b 1.49, m17.5a 1.49, m b 1.41, m17.3a 1.43, m b 1.33, m19.6
7a 1.77, m b 1.17, m29.8a 1.76, m b 1.15, m29.6a 1.93, m b 1.22, m34.4
836.536.535.9
91.26, m45.61.25, m46.01.41, m41.2
1035.135.035.3
11a 1.81, m b 1.75, m25.2a 1.84, m b 1.79, m25.31.86, m1.83, m23.6
125.26, m121.85.27, m122.05.18, m119.0
13137.0136.9137.6
141.14, m55.31.14, m55.41.13, t (4.0)54.6
15a 1.46, m b 1.28, m26.5a 1.46, m b 1.29, m26.4a 1.56, m b 1.21, m28.9
16a 1.73, m b 1.68, m32.7a 1.75, m b 1.68, m32.7a 1.85, m b 1.70, m33.6
173.07, t (5.9)54.93.08, t (5.9)54.93.08, t (5.9)54.8
18169.9169.9169.6
190.77, s16.00.84, s22.30.96, s20.0
200.86, s28.40.8, s28.50.99, s27.8
210.97, s27.00.99, s27.10.83, s23.8
220.90, s22.90.92, s22.70.95, s26.3
231.61, s22.91.61, s22.91.63, d (1.7)23.4
), ArticleFig(id=1200470902451335846, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, language=CN, label=Table 2, caption=

1H (500 MHz) and 13C (125 MHz) NMR data of compounds 1-3 in DMSO-d6

, figureFileSmall=null, figureFileBig=null, tableContent=
No.123
δH mult. (J in Hz)δCδH mult. (J in Hz)δCδH mult. (J in Hz)δC
11.30, m34.6a 1.78, m b 0.96, m29.2a 1.92, m b 1.46, m33.1
21.46, m27.9a 1.80, m b 1.40, m25.9a 2.65, ddd (15.6, 9.0, 5.5) b 2.27, ddd (15.6, 9.0, 5.5)33.7
32.97, m77.13.19, m73.7218.0
439.337.946.5
51.41, m43.01.92, m36.11.90, m47.8
6a 1.58, m b 1.49, m17.5a 1.49, m b 1.41, m17.3a 1.43, m b 1.33, m19.6
7a 1.77, m b 1.17, m29.8a 1.76, m b 1.15, m29.6a 1.93, m b 1.22, m34.4
836.536.535.9
91.26, m45.61.25, m46.01.41, m41.2
1035.135.035.3
11a 1.81, m b 1.75, m25.2a 1.84, m b 1.79, m25.31.86, m1.83, m23.6
125.26, m121.85.27, m122.05.18, m119.0
13137.0136.9137.6
141.14, m55.31.14, m55.41.13, t (4.0)54.6
15a 1.46, m b 1.28, m26.5a 1.46, m b 1.29, m26.4a 1.56, m b 1.21, m28.9
16a 1.73, m b 1.68, m32.7a 1.75, m b 1.68, m32.7a 1.85, m b 1.70, m33.6
173.07, t (5.9)54.93.08, t (5.9)54.93.08, t (5.9)54.8
18169.9169.9169.6
190.77, s16.00.84, s22.30.96, s20.0
200.86, s28.40.8, s28.50.99, s27.8
210.97, s27.00.99, s27.10.83, s23.8
220.90, s22.90.92, s22.70.95, s26.3
231.61, s22.91.61, s22.91.63, d (1.7)23.4
), ArticleFig(id=1200470902589747881, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
No.456
δH mult. (J in Hz)δCδH mult. (J in Hz)δCδH mult. (J in Hz)δC
11.31, m34.61.79, m 0.96, m29.11.91, m 1.47, m33.2
21.46, m27.91.80, m 1.40, m25.82.64, m 2.27, m33.8
32.97, dd (10.7, 5.0)77.23.19, m73.7218.1
439.337.946.6
51.41, m43.01.92, dd (1.8, 7.6)36.11.90, m47.8
61.58, m 1.50, m17.51.50, m 1.45, m17.31.46, m 1.31, m19.6
71.72, m 1.20, m29.91.70, m 1.20, m29.61.90, m 1.27, m34.5
836.536.636.0
91.26, m45.71.25, dd (12.4, 4.8)46.11.39, dd (11.1, 6.1)41.4
1035.135.035.3
111.81, m 1.75, m25.21.87, m 1.78, m25.31.87, m23.7
125.25, d (4.9)121.75.27, m122.05.18, m119.0
13137.2137.0137.8
141.18, m55.01.18, m55.01.18, m54.4
151.49, m 1.27, m26.11.47, m 1.26, m25.81.59, m 1.18, m28.3
161.70, m 1.51, m35.91.70, m 1.56, m35.61.72, m 1.67, m36.7
173.73, s70.83.85, dd (6.7, 4.7)70.23.87, t (5.4)70.2
18176.3175.7175.9
190.76, s16.00.84, s22.30.96, s20.0
200.85, s28.40.8, s28.50.99, s27.8
210.96, s27.10.98, s27.10.83, s23.9
220.9, s22.90.92, s22.70.95, s26.3
231.59, s22.81.60, d (1.8)22.71.61, s23.3
), ArticleFig(id=1200470902702994095, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200394153394819631, language=CN, label=Table 3, caption=

1H (500 MHz) and 13C (125 MHz) NMR data of compounds 4-6 in DMSO-d6

, figureFileSmall=null, figureFileBig=null, tableContent=
No.456
δH mult. (J in Hz)δCδH mult. (J in Hz)δCδH mult. (J in Hz)δC
11.31, m34.61.79, m 0.96, m29.11.91, m 1.47, m33.2
21.46, m27.91.80, m 1.40, m25.82.64, m 2.27, m33.8
32.97, dd (10.7, 5.0)77.23.19, m73.7218.1
439.337.946.6
51.41, m43.01.92, dd (1.8, 7.6)36.11.90, m47.8
61.58, m 1.50, m17.51.50, m 1.45, m17.31.46, m 1.31, m19.6
71.72, m 1.20, m29.91.70, m 1.20, m29.61.90, m 1.27, m34.5
836.536.636.0
91.26, m45.71.25, dd (12.4, 4.8)46.11.39, dd (11.1, 6.1)41.4
1035.135.035.3
111.81, m 1.75, m25.21.87, m 1.78, m25.31.87, m23.7
125.25, d (4.9)121.75.27, m122.05.18, m119.0
13137.2137.0137.8
141.18, m55.01.18, m55.01.18, m54.4
151.49, m 1.27, m26.11.47, m 1.26, m25.81.59, m 1.18, m28.3
161.70, m 1.51, m35.91.70, m 1.56, m35.61.72, m 1.67, m36.7
173.73, s70.83.85, dd (6.7, 4.7)70.23.87, t (5.4)70.2
18176.3175.7175.9
190.76, s16.00.84, s22.30.96, s20.0
200.85, s28.40.8, s28.50.99, s27.8
210.96, s27.10.98, s27.10.83, s23.9
220.9, s22.90.92, s22.70.95, s26.3
231.59, s22.81.60, d (1.8)22.71.61, s23.3
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Brasilicardin A生物合成相关二萜合酶的异源表达及产物鉴定
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葛祥宇 1 , 周广鑫 1 , 熊娜 1 , 卢姿含 1 , 米芯雨 1 , 朱枝祥 1 , 刘晓 1 , 王晓晖 1 , 王娟 1, 2, * , 史社坡 1, *
药学学报 | 研究论文 2024,59(7): 2161-2170
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药学学报 | 研究论文 2024, 59(7): 2161-2170
Brasilicardin A生物合成相关二萜合酶的异源表达及产物鉴定
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葛祥宇1, 周广鑫1, 熊娜1, 卢姿含1, 米芯雨1, 朱枝祥1, 刘晓1, 王晓晖1, 王娟1, 2, * , 史社坡1, *
作者信息
  • 1.北京中医药大学, 中药现代研究中心, 北京 102488
  • 2.中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 北京 100050

通讯作者:

*王娟, Tel: 86-10-53911868, E-mail: ;
史社坡, E-mail:
Heterologous expression and product identification of diterpene synthase involved in the biosynthesis of brasilicardin A
Xiang-yu GE1, Guang-xin ZHOU1, Na XIONG1, Zi-han LU1, Xin-yu MI1, Zhi-xiang ZHU1, Xiao LIU1, Xiao-hui WANG1, Juan WANG1, 2, * , She-po SHI1, *
Affiliations
  • 1. Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 102488, China
  • 2. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
出版时间: 2024-07-12 doi: 10.16438/j.0513-4870.2024-0089
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摘要
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从致病性放线菌Nocardia brasiliensis IFM 0406中得到的二萜糖苷brasilicardin A, 因免疫抑制活性显著、毒性较低且作用机制独特而成为新型免疫抑制剂的候选分子。由于原菌株中产率低, 且结构复杂、化学合成困难, brasilicardin A及其类似物的获得成为该类新型免疫抑制剂的研究热点。根据目前报道的brasilicardin A可能的生物合成途径, 通过生物信息学分析生物合成相关二萜合酶, 定向从致病菌株N. brasiliensis IFM 0406中扩增可能合成brasilicardin A骨架的基因bra1~5, 在白色链霉菌Streptomyces albus R1中成功实现异源表达。通过液体发酵培养, 利用硅胶柱色谱及半制备液相色谱等方法进行化合物的分离纯化, 共分离到6个新brasilicardins化合物, 命名为brasilicardin H~M。利用脂多糖(LPS) 活化的小鼠原代巨噬细胞炎症模型对分离鉴定的brasilicardins进行了体外活性筛选, 结果显示, brasilicardin H~M具有较强抑制巨噬细胞释放一氧化氮(NO) 的作用, IC50分别为28.24 ± 3.70、37.44 ± 2.00、39.85 ± 4.02、26.77 ± 4.40、65.25 ± 1.48、15.24 ± 2.72 μmol·L-1 (阳性对照吲哚美辛IC50为34.28 ± 4.10 μmol·L-1), 表明6个化合物具有潜在的抗炎活性。本研究为brasilicardin A生物合成途径的彻底阐明提供了条件, 也为研究该类化合物的构效关系、药物研发等奠定了基础。

brasilicardin A  /  二萜合酶  /  异源表达  /  免疫抑制活性

Brasilicardin A, a diterpene glycoside isolated from pathogenic actinomycete Nocardia brasiliensis IFM 0406, has become a novel immunosuppressant candidate due to its significant immunosuppressive activity, low toxicity and unique mechanism of action. However, brasilicardin A and its analogues have become a research hotspot to the development of this promising immunosuppressant because of the low-yield production in the natural pathogenic producer and the synthetically challenging skeleton. According to the reported biosynthetic pathway of brasilicardin A, the function of involved diterpene synthase was analyzed by bioinformatics. Then the genes bra1-5 that synthesize the brasilicardin A skeleton were directionally amplified from the pathogenic strain N. brasiliensis IFM 0406, and heterologous expression was achieved successfully in Streptomyces albus R1. The compounds were isolated and purified by using various column chromatographies including silica gel column chromatography and semi-preparative HPLC. Six new brasilicardins were established and named brasilicardin H-M. The activity of brasilicardins was screened using lipopolysaccharide (LPS)-activated mouse primary macrophage inflammation model. Brasilicardin H-M exhibited good inhibitory activity on nitric oxide (NO) release with IC50 values of 28.24 ± 3.70, 37.44 ± 2.00, 39.85 ± 4.02, 26.77 ± 4.40, 65.25 ± 1.48 and 15.24 ± 2.72 μmol·L-1, respectively (indomethacin as the positive control with IC50 value of 34.28 ± 4.10 μmol·L-1). The results indicated that six compounds had potential anti-inflammatory activity. This study laid a foundation for the elucidation of the brasilicardin A biosynthetic pathway and evaluation of the structure-activity relationship as well as new drug developments.

brasilicardin A  /  diterpene synthase  /  heterologous expression  /  immunosuppressive activity
葛祥宇, 周广鑫, 熊娜, 卢姿含, 米芯雨, 朱枝祥, 刘晓, 王晓晖, 王娟, 史社坡. Brasilicardin A生物合成相关二萜合酶的异源表达及产物鉴定. 药学学报, 2024 , 59 (7) : 2161 -2170 . DOI: 10.16438/j.0513-4870.2024-0089
Xiang-yu GE, Guang-xin ZHOU, Na XIONG, Zi-han LU, Xin-yu MI, Zhi-xiang ZHU, Xiao LIU, Xiao-hui WANG, Juan WANG, She-po SHI. Heterologous expression and product identification of diterpene synthase involved in the biosynthesis of brasilicardin A[J]. Acta Pharmaceutica Sinica, 2024 , 59 (7) : 2161 -2170 . DOI: 10.16438/j.0513-4870.2024-0089
正文
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Brasilicardin A是从致病性放线菌Nocardia brasiliensis IFM 0406中发现的具有显著免疫抑制作用的二萜类化合物, 其独特的作用机制开辟了免疫抑制剂研究新领域[1, 2]。但brasilicardin A的原始菌株的致病性和brasilicardin A合成的挑战性使得brasilicardin A及其类似物的高效获得还存在一定的瓶颈[3, 4]。二萜合酶(diterpene synthases, DTSs) 是二萜类化合物结构合成的关键酶, 起到环化、结构多样性的作用。Brasilicardin A完整的生物合成基因簇尚未阐明, 对已报道的brasilicardin A可能的生物合成途径相关基因进行生物信息学分析, brasilicardin A生物合成途径相关基因簇由鲨烯环氧化酶(bra5)、二萜环合酶(bra4) 以及其他后修饰基因等在内的共12个基因(bra0~11) 构成(图 1)。其中Bra4能环合生成brasilicardin A的骨架, 经过系统进化分析发现Bra4属于非典型Ⅱ型二萜合酶, 序列GC含量高, 其缺少典型二萜合酶的DXDD保守结构域但含有特异的E/DSAE/N结构域, 且利用以epoxy-GGPP (geranylgeranyl pyrophosphate) 为底物而非传统的GGPP, 是目前很罕见的一类细菌二萜合酶[5], Bra4独特的酶结构和催化机制可以合成特异的反式/顺式/反式三环二萜, 但Bra4的直接环化产物目前尚未解析, 其免疫抑制活性也并不明确。
异源表达宿主能弥补原始菌株生长环境苛刻且生长速率缓慢、分子生物学工具匮乏、产量低、菌株致病性等缺点[6], 在活性天然产物产量的提高、生物合成基因簇的鉴定、新颖化合物的发现以及生物合成途径的改造实现结构的衍生化方面越来越受到科研工作者的重视[7]。例如: 合成抗肿瘤药物的重要先导化合物星形孢菌素(staurosporine) 在异源宿主Streptomyces albus J1074中产量提高30倍[8]; 在Myxococcus xanthus DK1622宿主菌中异源表达了vioprolide生物合成基因簇并实现了vioprolides的高产[9]; 在Streptomyces coelicolor中异源表达来自“沉默”的生物合成基因簇tar揭示了两种新型脂肽类抗生素taromycin A2和B3[10, 11]; 在Aspergillus oryzae中异源表达隐秘基因簇cle, 获得了二萜化合物chevalone E及其氧化类似物, 替换其中萜烯环化酶基因产生了sartorypyrone D[12]。抗癌药物前体吉马烯A (germacrene A) 的生物合成路径在解脂耶氏酵母中重编程提高产量至39 g·L-1 [13]。文多灵(vindoline) 和长春花碱(catharanthine) 也在酵母中实现从头合成[14]。目前多种异源表达宿主已凭借自身独特的优势在实现多种类型化合物的生物合成和新颖活性分子的发现方面取得了巨大的成果。
因此利用基因簇中关键的二萜环合酶在其他同源微生物中外源合成brasilicardins, 为解决目前brasilicardin A及类似物稀缺、化合物资源限制药物研发的瓶颈提供物质基础。考虑到Bra4直接环化产物不稳定难以检测到, 异源表达时加入后修饰基因bra3, 同时基于链霉菌宿主有利于异源表达高GC含量的基因组DNA的优势, 本课题将合成brasilicardin A二萜母核基因bra1~5[5]在链霉菌宿主Streptomyces albus R1中异源表达, 从其发酵产物中分离鉴定了6个新化合物brasilicardin H~M, 活性检测结果显示brasilicardin H~M具有较强抑制脂多糖(LPS) 诱导的小鼠原代巨噬细胞释放一氧化氮(NO) 的作用, 为后续阐明brasilicardin A的生物合成途径以及筛选活性显著的brasilicardins活性分子奠定了基础。
材料与方法
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试剂    巴西诺卡菌N. brasiliensis IFM 0406基因组DNA购自千叶大学真菌医学研究中心; 硫酸阿布拉霉素(apramycin sulfate, Apr)、卡那霉素(kanamycin, Kana)、氯霉素(chloramphenicol, Chl) 以及萘啶酮酸均购自生工生物工程(上海) 股份有限公司; DNA连接酶和Pfu高保真DNA聚合酶购自北京全式金生物科技有限公司; 限制性内切酶购自北京宝日医生物科技有限公司; 乙酸乙酯、无水乙醇、甲醇等分析纯试剂购自西陇科学股份有限公司; 甲酸、乙酸采购自天津市大茂化学试剂厂。
仪器    R-210旋蒸仪购自瑞士BUCHI公司; UVP凝胶成像仪、凝胶电泳仪购自美国BioRad公司; NanoDrop 2000C分光光度计购自美国ThermoFisher Scientific公司; 化合物分析、精确分子量以及二级质谱数据采集采用高效液相离子阱飞行时间质谱分析系统(UFLC SIL-20AC自动进样器, CTO-20AC柱温箱, SPD M20A紫外检测器, LC-20ADXR泵, IT-TOF-MS配备ESI离子源) 购自日本Shimadzu公司; 化合物的制备采用Waters 2998半制备型高效液相色谱仪以及SunFireTM C18半制备柱(150 mm×10 mm, 5 μm), 美国Waters公司; 化合物结构解析采用美国Varian 500M核磁共振仪。摇床购自北京京创泰宁伟业科技发展有限公司; 电转仪、梯度PCR仪、高速离心机购于德国Eppendorf公司; 恒温培养箱购自上海精宏实验设备有限公司。
实验动物    雄性无特定病原体(specific pathogen free, SPF) 级C57BL/6小鼠15只, 8周龄, 体重20~25 g, 购自斯贝福(北京) 生物技术有限公司(许可证号: SCXK (京) 2019-0010), 饲养于北京中医药大学实验动物中心屏障环境, 室温20~24 ℃, 相对湿度45%~65%。本研究方案通过北京中医药大学实验动物伦理委员会审核。
菌株、质粒和引物    本文所用菌株、质粒和引物见表 1 (所用引物均由深圳华大基因股份有限公司合成)。
基因的克隆与整合型质粒的构建    以巴西诺卡菌N. brasiliensis IFM 0406基因组DNA为模板, Primer 1和Primer 2为引物, PCR扩增后可得到6 kb大小的含bra1~5基因片段, 基因测序正确后, 利用Nde Ⅰ和Xba Ⅰ限制性核酸内切酶位点将该片段连接到整合型载体pIB139上, 得到含有bra1~5基因的目的质粒pGXY1。
接合转移与接合子的筛选    首先将E.coli ET12567/pUZ8002制备电转感受态细胞, 将用于接合转移的质粒pGXY1通过电转转化的方法导入E. coli ET12567/pUZ8002中, 筛选阳性克隆。将S. albus R1的甘油菌涂布在新鲜的SFM固体板[15]上, 30 ℃培养箱倒置孵育3天以获得孢子, 将携带质粒pGXY1的E. coli ET12567/pUZ8002在37 ℃恒温摇床以转速180 r·min-1振荡培养过夜, 次日按照1%的比例接种到20 mL含有50 μg·mL-1 Kana、25 μg·mL-1 Chl和50 μg·mL-1 Apr的LB (Luria-Bertani)[16]液体培养基中, 37 ℃恒温摇床以转速180 r·min-1振荡培养至OD600值0.2~0.6, 7 600 r·min-1离心10 min收集菌体, 用20 mL 2×YT培养基[15]漂洗2次后, 重悬于300 μL 2×YT培养基中, 作为供体细胞; 用接种环刮取在SFM固体板上孵育的S. albus R1的孢子, 用2×YT培养基漂洗去掉菌丝后, 将孢子重悬于200 μL 2×YT培养基中, 50 ℃热激10 min后, 室温放置10 min使孢子萌发, 作为受体; 将供体细胞与受体细胞混合, 在含CaCl2和MgCl2的ISP4固体板[17]上30 ℃孵育16~20 h后用50 μg·mL-1 Apr及40 μg·mL-1的萘啶酮酸覆盖, 待ISP4平板在无菌超净台静置晾干后, 倒置培养5天可以观察到接合子。将接合子再次接种到含50 μg·mL-1硫酸阿布拉霉素及40 μg·mL-1的萘啶酮酸的新鲜SFM培养皿上, 倒置培养于30 ℃恒温培养箱。3~5天后可观察到链霉菌菌丝体和孢子的生成, 取适量孢子加少量DMSO涡旋2 min后作为模板用Primer 3和Primer 4引物PCR扩增质粒载体上的基因, 确保质粒成功整合到链霉菌宿主的染色体上, 得到相应突变株S. albus R1/pGXY1, 命名为GXY01。
链霉菌的培养与保藏    使用无菌接种环将菌株GXY01的孢子均匀涂布在SFM固体培养基表面, 将其放置在30 ℃恒温培养箱倒置培养5天, 当观察到链霉菌的白色孢子时, 用无菌接种环从培养基表面轻轻刮取孢子, 重悬于装有20%无菌甘油的冻存管中, 振荡混匀后液氮速冻于-80 ℃保藏。
链霉菌的发酵和样品制备    链霉菌宿主发酵采用A液体培养基[18]。将所得突变株和相应的对照菌株接种于无菌的含有50 μg·mL-1 Apr的A液体培养基中, 在30 ℃恒温摇床以转速180 r·min-1振荡培养1天后得到发酵种子液。以5%比例将种子液均匀接种于无菌的含有50 μg·mL-1 Apr的A液体培养基中, 在30 ℃恒温摇床以转速180 r·min-1振荡培养5天后得到发酵液。发酵完成后, 4 500 r·min-1离心20 min将菌体和菌液分离, 分别用乙酸乙酯萃取3次获得发酵产物萃取液, 减压浓缩后用1 mL甲醇溶解, 12 000 r·min-1低温离心15 min, 取上清用于HPLC-IT-TOF检测分析的发酵样品。
发酵产物的分离纯化    选取突变株GXY01为发酵菌株, 用无菌接种环将GXY01的孢子接种于无菌的含有50 μg·mL-1 Apr的50 mL A液体培养基中, 于30 ℃振荡培养1天获得种子液。配制15 L A液体培养基, 以5%比例将种子液均匀接种于无菌的含有50 μg·mL-1 Apr的A液体培养基中, 在30 ℃恒温摇床以转速180 r·min-1振荡培养5天后得到发酵液。发酵完成后, 4 500 r·min-1离心20 min将菌体和菌液分离, 分别用乙酸乙酯萃取3次获得发酵产物萃取液, 减压浓缩后获得发酵粗产物2 g。将所有粗产物上样于凝胶分离柱, 采用甲醇-0.1%甲酸水等度洗脱, 共收集12瓶馏分, 编号依次为Fr.1~Fr.12。Fr.8 (203.7 mg) 通过prep-HPLC制备(乙腈-0.2%乙酸水60∶40, 流速: 3 mL·min-1), 得到馏分Fr.8.1~Fr.8.4。Fr.8.2 (10.7 mg) 通过prep-HPLC制备(甲醇-0.2%乙酸水70∶30, 流速: 3 mL·min-1), 得到化合物brasilicardin L (tR = 25 min, 3.1 mg) 和化合物brasilicardin K (tR = 45 min, 3.9 mg)。Fr.8.4 (107.7 mg) 通过prep-HPLC制备(甲醇-0.2%乙酸水75∶25, 流速: 3 mL·min-1), 得到化合物brasilicardin M (tR = 30 min, 4.7 mg)。Fr.11 (658.7 mg) 通过prep-HPLC制备梯度洗脱(A: 0.2%乙酸水; B: 乙腈; 流速: 3 mL·min-1), 梯度洗脱程序为0~20 min 5%~95% B, 20~26 min 95% B, 26~26.10 min 95%~5% B, 26.10~31 min 5% B, 得到馏分Fr.11.1~Fr.11.4。Fr.11.2 (225.3 mg) 通过prep-HPLC制备(乙腈-0.2%乙酸水70∶30, 流速: 3 mL·min-1), 得到馏分Fr.11.2.1~Fr.11.2.9。Fr.11.2.5 (14 mg) 通过prep-HPLC制备(甲醇-0.2%乙酸水58∶42, 流速: 3 mL·min-1), 得到化合物brasilicardin I (tR = 18 min, 5.0 mg)。Fr.11.2.7 (8 mg) 通过prep-HPLC制备(甲醇-0.2%乙酸水65∶35, 流速: 3 mL·min-1), 得到brasilicardin H (tR = 19 min, 4.8 mg)。Fr.11.2.9 (32 mg) 通过prep-HPLC制备(甲醇-0.2%乙酸水65∶35, 流速: 3 mL·min-1), 得到化合物brasilicardin J (tR =28 min, 14.0 mg)。
底物饲喂    链霉菌宿主菌株S. albus R1接种于5 mL A液体培养基中, 同时设置空白对照组(即5 mL无S. albus R1的A液体培养基) 在30 ℃恒温摇床以转速180 r·min-1振荡培养1天后, 分别加入5 μL 25 mmol·L-1的化合物brasilicardin H~M作为底物饲喂到宿主菌S. albus R1中, 并同时在A液体培养基中加入5 μL 25 mmol·L-1的化合物brasilicardin H~M作为空白对照组。在30 ℃恒温摇床以转速180 r·min-1振荡培养5天后得到发酵液。发酵完成后, 用乙酸乙酯萃取3次获得发酵产物萃取液, 减压浓缩后用500 μL甲醇溶解, 12 000 r·min-1低温离心15 min, 取上清用于HPLC-IT-TOF检测分析的发酵样品。
色谱条件    产物检测条件采用Agilent Extend-C18色谱柱(250 mm×4.6 mm, 5 μm); 梯度洗脱采用溶剂A (0.1%甲酸水) 和溶剂B (乙腈), 梯度洗脱程序为0~2 min 5% B, 2~22 min 5%~95% B, 22~28 min 95% B, 28~28.10 min 95%~5% B, 28.10~33 min 5% B。进样量20 μL, 流速1.0 mL·min-1, 柱温40 ℃。底物饲喂检测条件采用Agilent Extend-C18色谱柱(250 mm×4.6 mm, 5 μm); 梯度洗脱采用溶剂A (0.1%甲酸水) 和溶剂B (甲醇), 梯度洗脱程序为0~5 min 50% B; 5~9 min 50%~70% B; 9~14 min 70% B; 14~24 min 70%~90% B; 24~25 min 90%~100% B; 25~30 min 100% B; 30~31 min 100%~50% B; 31~36 min 50% B。进样量20 μL, 流速1.0 mL·min-1, 柱温40 ℃。
质谱条件    离子源为电喷雾离子源(electron spray ionization, ESI), 扫描模式为正、负离子同时扫描并自动触发多级质谱, MS1的质量扫描范围m/z 100~1 000, MS2的质量扫描范围m/z 50~800。喷雾室电压4.5~3.5 kV; 雾化气(N2) 流速: 1.5 L·min-1; 干燥气体压力100 MPa; 检测电压1.5 kV; CDL温度200 ℃; 碰撞诱导解离(CID) 能量为70%。
ECD计算    首先使用分子力场MMFF94进行构象搜索。然后, 在Molclus (版本1.9.9.4) 软件中基于xtb程序使用GFN-xTB方法进行结构优化。选择相对能量低于5 kcal·mol-1的构象通过Gaussian 09软件气相条件下在B3LYP/6-31G (d) 水平进行进一步优化。溶剂化能通过自洽反应场(SCRF) 使用SMD连续溶剂化模型在M052X/6-31G (d) 水平下评估, 溶剂为甲醇。单点能在M062X/def2-TZVP水平下计算得到。最后, 得到每个结构的最低能量构象。然后选择玻尔兹曼系数大于1%的构象进行ECD计算。使用含时密度泛函理论(TD-DFT) 在Cam-B3LYP/6-311G (d, p) 水平下进行ECD计算。ECD光谱由最低能量构象的玻尔兹曼加权生成。
生物活性检测    收集雄性C57BL/6小鼠原代巨噬细胞, 将细胞数稀释到每毫升1.5×106个, 接种100 µL到96孔板中, 每孔1.5×104个, 细胞培养箱孵育过夜。单体化合物配制成25 mmol·L-1浓度母液, 然后用培养基稀释后加入96孔培养板至终浓度为100、20、4 μmol·L-1, 培养箱孵育15 min, 再加入LPS至终质量浓度为1 μg·mL-1, 继续孵育24 h。从96孔板各孔中吸取100 µL上清至酶标板内, 每孔加入50 µL Griess R1, 室温反应5 min, 再加入50 µL Griess R2, 室温反应5 min, 540 nm测定吸光度, 并计算各孔NO浓度和化合物对NO分泌的抑制率, 抑制率 > 50%的计算半数抑制浓度(IC50)。在上述96孔板中加入10 µL CCK-8, 继续置于细胞培养箱内孵育90 min后测定450 nm吸光度, 并计算化合物对原代巨噬细胞生长的抑制率。
结果与分析
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1 Brasilicardin A生物合成基因簇中二萜母核基因bra1~5的异源表达
根据目前已知的brasilicardin A的生物合成途径可知, N. brasiliensis IFM 0406基因组中bra1~5负责合成brasilicardin A母核。将其与pIB139载体连接后, 得到了含有基因bra1~5的质粒pGXY1。通过接合转移的方法, 将质粒pGXY1导入白色链霉菌S. albus R1中, 将获得的接合子通过引物Primer 3和Primer 4进行PCR验证, 验证成功的接合子命名为GXY01。利用HPLC-IT-TOF对重组菌株GXY01的发酵液进行检测。结果显示, 与对照菌株相比, 重组菌株中多出质荷比m/z 378.299 9 [M+H]+、376.283 5 [M+H]+、377.269 1 [M-H]-、375.253 8 [M-H]-的准分子离子峰(图 2)。经过分子式预测可能为二萜母核相关的化合物。
2 目标化合物的结构解析
从重组菌株GXY01发酵液中分离得到的化合物brasilicardin H~M均为二萜类化合物(图 3、4), 分子结构由罕见的反式/顺式/反式全氢菲母核和氨基酸部分组成。
化合物1为白色粉末, HR-ESI-MS中给出准分子离子峰[M+H]+: m/z 378.299 9 (calcd. for 378.300 8), 提示分子式为C23H39NO3, 计算不饱和度为5。
1H NMR (DMSO-d6, 500 MHz) 谱图中给出一个烯氢信号: δH 5.26 (1H, m, H-12); 一个连氮次甲基氢信号: δH 3.07 (1H, t, J = 5.9 Hz, H-17); 一个连氧次甲基氢信号: δH 2.97 (1H, m, H-3) 以及五个甲基氢信号: δH 1.61 (3H, s, H-23)、0.97 (3H, s, H-21)、0.90 (3H, s, H-22)、0.86 (3H, s, H-20)、0.77 (3H, s, H-19)。
13C NMR (DMSO-d6, 125 MHz) 谱图中给出23个碳信号, 包括一个羧基碳信号: δC 169.9 (C-18); 一组双键碳信号: δC 137.0 (C-13)、121.8 (C-12); 一个连氧碳信号: δC 77.1 (C-3); 一个连氮碳信号: δC 54.9 (C-17); 五个甲基碳信号: δC 28.4 (C-20)、27.0 (C-21)、22.9 (C-22)、22.9 (C-23)、16.0 (C-19)。综合1H NMR、13C NMR数据(表 2) 推测化合物1存在一个二萜母核。
通过2D NMR实验进一步确定了化合物1的结构。HMBC谱图中, H-1与C-3/C-5相关; H-2与C-4/C-10相关; H-6与C-8/C-10相关; H-7与C-5/C-9相关; H-9与C-5/C-14相关; H-11与C-8/C-13相关; H-12与C-9/C-14相关; H-14与C-16相关; H-15与C-8相关; H-17与C-15相关; H3-19/H3-20与C-3/C-5相关; H3-21与C-1/C-9相关; H3-22与C-7/C-14相关; H3-23与C-12/C-14相关(图 3), 确定了化合物1的平面结构。
根据brasilicardins生物合成途径分析可知该类型化合物的二萜母核构型十分保守, 同时对比brasilicardin E[4]的核磁数据, 发现化合物1相比brasilicardin E在2位少一个羟基, 16位少一个甲氧基, 结合ROESY谱图中H-3与H-5相关, H3-20/H3-22与H-6a相关, H-7a与H-14和H-5相关、H-9与H-15a相关。以上结果表明, H-11b和H3-19为α取向, H-3、H-5、H-6a、H-14和H3-20为β取向。因此化合物1的绝对构型为3R, 5S, 8S, 9S, 10S, 14S, 17S, 命名为brasilicardin H。
化合物2为白色粉末, HR-ESI-MS中给出准分子离子峰[M+H]+: m/z 378.300 2 (calcd. for 378.300 8), 提示分子式为C23H39NO3, 计算不饱和度为5。比较二者的NMR数据(表 2) 发现化合物2的数据与1十分相似, 两者区别在化合物2中1位至5位的碳谱数据向高场位移, 提示其可能为1的差向异构体。ROESY谱图中给出H-3与H3-21相关信号, 表明H-3为α取向。综上所述, 确定化合物2的3位绝对构型为S构型, 其他部分结构与化合物1一致, 命名为brasilicardin I。
化合物3为白色粉末, HR-ESI-MS中给出准分子离子峰[M+H]+: m/z 376.283 5 (calcd. for 376.285 2), 提示分子式为C23H37NO3, 计算不饱和度为6。综合1H NMR和13C NMR数据(表 2) 发现化合物3的数据与1十分相似, 两者区别仅在1的3位氢信号消失, 碳信号向低场位移(δC 218.0), 推测3-OH在3中被氧化为羰基。HMBC谱图中给出了H-1/H-5与羰基碳C-3存在相关, 进一步证明了以上推断。通过详细的HMBC与HSQC信号分析(图 3) 确定化合物3的其他部分结构与1一致。此外, 化合物3的实验ECD曲线与计算出的模型分子3a(17S) 的ECD光谱曲线吻合较好(图 5A)。确定化合物3的结构与3a一致, 即化合物3在17位绝对构型与化合物1、2均为S构型。综上所述化合物3被命名为brasilicardin J。
化合物4为白色粉末, HR-ESI-MS中给出准分子离子峰[M-H]-: m/z 377.269 1 (calcd. for 377.269 2), 提示分子式为C23H38O4, 计算不饱和度为5。综合1H NMR和13C NMR数据(表 3) 发现化合物4的数据与1十分相似, 两者区别仅在4中17位的氢、碳信号向低场位移(δH 3.73, δC 70.8), 推断17-OH代替了1中的17-NH2。HMBC谱图中给出了H-17与C-15相关, H-15与C-13相关, 进一步证明了以上推断。通过计算与实验ECD曲线对比确定化合物6的17位绝对构型为R构型, 结合详细的核磁数据分析(图 3) 确定化合物4的17位绝对构型与6一致, 确定4的17位绝对构型为R构型。通过详细的HMBC与HSQC信号分析(图 3) 确定化合物4的其他部分结构与1一致。故化合物4被鉴定为brasilicardin K。
化合物5为白色粉末, HR-ESI-MS中给出准分子离子峰[M-H]-: m/z 377.269 2 (calcd. for 377.269 2), 提示分子式为C23H38O4, 计算不饱和度为5。比较二者的NMR数据(表 3) 发现化合物5的数据与4十分相似, 两者区别在化合物5中1位至5位的碳谱数据向高场位移, 提示其可能为4的差向异构体。ROESY谱图中给出H-3与H3-21相关信号, 表明H-3为α取向, 确定5的3位绝对构型为S构型, 其他部分结构与4一致。故化合物5被鉴定为brasilicardin L。
化合物6为白色粉末, HR-ESI-MS中给出准分子离子峰[M-H]-: m/z 375.253 8 (calcd. for 375.253 5), 提示分子式为C23H36O4, 计算不饱和度为6。综合1H NMR, 13C NMR数据(表 3) 发现化合物6的数据与4十分相似, 两者区别仅在6的3位氢信号消失, 碳信号向低场位移(δC 218.1), 推测3-OH在6中被氧化为羰基。HMBC谱图中给出了H-1/H-5与羰基碳C-3存在相关, 进一步证明了以上推断。通过详细的HMBC与HSQC信号分析(图 3) 确定化合物6的其他部分结构与4一致。化合物6的实验ECD曲线与模型分子6b (17R) 的计算ECD曲线吻合较好(图 5B)。确定化合物6的结构与6b一致, 即化合物6的17位绝对构型为R构型。故化合物6被鉴定为brasilicardin M。
3 活性检测
对上述6个化合物进行活性筛选, 结果显示, brasilicardin H~M具有较强抑制LPS诱导的小鼠原代巨噬细胞释放NO的作用, IC50分别为28.24 ± 3.70、37.44 ± 2.00、39.85 ± 4.02、26.77 ± 4.40、65.25 ± 1.48、15.24 ± 2.72 μmol·L-1 (阳性对照吲哚美辛IC50为34.28 ± 4.10 μmol·L-1), 表明6个化合物均具有潜在的抗炎活性。
4 生物合成途径
将bra1~5在链霉菌宿主S. albus R1中异源表达, 不仅得到brasilicardin H, 还分离得到了其他5个新颖的类似物brasilicardin I~M。根据brasilicardin I~M的结构特点, 推测这5个化合物可能是由宿主S. albus R1中的内源性短链脱氢酶(short chain dehydrogenase) 介导的3位羟基的构型翻转[19]。因此, 本文分别考察了宿主S. albus R1对brasilicardin I~M的转化情况, 通过对转化产物的LC-MS分析(图 6A~F), 证实了上述推测。在野生型S. albus R1转化体系中, brasilicardin H (1) 可以转化成brasilicardin J (3) (图 6A), brasilicardin J (3) 能转化成brasilicardin I (2) (图 6B), 而brasilicardin I (2) 还能再转化成brasilicardin J (3) (图 6C)。同样, 野生型S. albus R1能将brasilicardin K (4) 转化成brasilicardin M (6) (图 6D), brasilicardin M (6) 能转化成brasilicardin L (5) (图 6E), 同时也能将brasilicardin L (5) 再转化成brasilicardin M (6) (图 6F)。因此, 推测brasilicardin H~M的生成途径如图 6G所示, 其中化合物brasilicardin K (4) 中的C16羰基转化为羟基的生成机制同样也有可能是宿主内源酶的作用。
讨论
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Bra4是目前发现的罕见的非典型二萜合酶, 但其重组蛋白难获得且催化底物难合成, Bra4的功能尚未解析。而尝试在菌株中特异敲除bra3后, 也未检测到Bra4的环化产物及任何的brasilicardins。因此本研究选择通过在链霉菌底盘宿主S. albus R1中异源表达包括萜类环合酶基因bra4在内的形成brasilicardin A二萜母核的基因bra1~5, 分离鉴定了brasilicardin A生物合成途径中具有反式/顺式/反式全氢菲母核和氨基酸部分在内的骨架化合物brasilicardin H, 补充目前brasilicardin A的生物合成途径的同时也为后续brasilicardin A的生物合成途径的阐明奠定了基础; 而底盘宿主内源基因的修饰产生许多衍生物的报道已有很多, 这极大地增大了化合物的多样性, 在本研究中由于链霉菌底盘宿主S. albus R1中内源基因对brasilicardin H的结构修饰, 分离奠定出5个新brasilicardins化合物brasilicardin I~M。
在brasilicardin H~M母核的基础上, 后续还可以利用积累的后修饰功能的系列催化元件库, 如引入糖基转移酶、羟化酶、氧甲基转移酶、异戊烯基转移酶以及氧化酶等其他修饰基因元件, 通过组合合成进一步增加brasilicardins的多样性用于药理活性筛选。另一方面, 由于目前异源表达后化合物的产率较小, 后续将通过优化培养基、增加生物合成前体的供应[20]、删除宿主基因组中可缺失的基因簇[21, 22]等多种方式进一步提高brasilicardin H~M的产量。
从brasilicardin A显著的免疫抑制活性发现以来, 如何高效获得brasilicardin A及发现更多其类似物一直是研究热点, 本研究旨在补充了brasilicardin A生物合成途径中的中间体同时也发现多个brasilicardins化合物, 但药理活性远远低于brasilicardin A, 说明brasilicardin A母核上的其他基团十分重要, 在后续研究中将基于目前研究成果阐明brasilicardin A的生物途径中后续相关基因; 也将基于目前分离到的化合物, 筛选各种来源的基因以期通过组合合成的方式获得多个brasilicardins化合物, 丰富brasilicardins这类化合物。
作者贡献: 葛祥宇负责基因bra1~5的克隆和异源表达、化合物的分离纯化以及论文初稿的撰写; 周广鑫负责化合物brasilicardin H~M谱图整理; 熊娜和米芯雨负责异源表达菌株GXY01的发酵与发酵液的萃取; 卢姿含负责化合物brasilicardin H~M的活性检测; 朱枝祥、刘晓和王晓晖提供实验指导以及论文修改; 王娟和史社坡负责实验方案设计、实验技术指导、论文撰写及修改。
利益冲突: 所有作者均声明研究内容无任何利益冲突。
基金
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  • 国家自然科学基金项目(81903495)
  • 天然药物活性物质与功能国家重点实验室开放课题(GTZK202203)
文献
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参考文献 引证文献
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2024年第59卷第7期
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doi: 10.16438/j.0513-4870.2024-0089
  • 接收时间：2024-01-27
  • 首发时间：2025-11-26
  • 出版时间：2024-07-12
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  • 收稿日期：2024-01-27
  • 修回日期：2024-05-07
基金
国家自然科学基金项目(81903495)
天然药物活性物质与功能国家重点实验室开放课题(GTZK202203)
作者信息
    1.北京中医药大学, 中药现代研究中心, 北京 102488
    2.中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 北京 100050

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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
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红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
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光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
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