合成生物学 ›› 2021, Vol. 2 ›› Issue (5): 837-849.DOI: 10.12211/2096-8280.2021-024
• 研究论文 • 上一篇
周海波, 申琪瑶, 陈汉娜, 王宗杰, 李越中, 张友明, 卞小莹
收稿日期:
2021-02-20
修回日期:
2021-04-06
出版日期:
2021-11-19
发布日期:
2021-11-19
通讯作者:
张友明,卞小莹
作者简介:
基金资助:
Haibo ZHOU, Qiyao SHEN, Hanna CHEN, Zongjie WANG, Yuezhong LI, Youming ZHANG, Xiaoying BIAN
Received:
2021-02-20
Revised:
2021-04-06
Online:
2021-11-19
Published:
2021-11-19
Contact:
Youming ZHANG, Xiaoying BIAN
摘要:
黏细菌是天然产物的重要来源。纤维堆囊菌So0157-2是抗癌药物埃博霉素的产生菌,并且是已知基因组最大的原核生物。生物信息学分析发现该菌一共含有35个次级代谢产物生物合成基因簇,除了已知的埃博霉素基因簇和其他两个萜类化合物生物合成基因簇与已知基因簇的相似度为100%之外,其他基因簇与已知化合物基因簇相似度均较低,其中包括17个聚酮合酶(polyketide synthase,PKS)、非核糖体肽合成酶(nonribosomal peptide synthetase,NRPS)及其杂合的基因簇。由于纤维堆囊菌So0157-2生长缓慢、培养困难且难以在本源菌中进行遗传改造。因此,将其生物合成基因簇转移到简单宿主中,利用异源表达策略是挖掘该菌中新颖天然产物的一个有效途径。本文利用直接克隆技术从纤维堆囊菌So0157-2基因组DNA中克隆了1个包含NRPS和PKS结构域的基因簇BGC18,将其转移至伯克氏菌DSM 7029中进行异源表达。通过液质联用分析,色谱柱靶向分离纯化,进而通过NMR结构鉴定和Marfey反应确定了3个化合物分别为Cyclo(N-Me-L-Leu-L-Val)(1)、Cyclo(N-Me-L-Leu-L-Leu)(2)、Cyclo(N-Me-L-Leu-L-Ile)(3)。化合物结构的多样性来源于第1个腺苷化结构域对底物识别的宽泛性(Val/Leu/Ile)。生物合成途径分析推测由于缺少硫醇化结构域导致PKS模块被跳过,从而只获得了NRPS指导合成的环二肽产物1~3,这可能是细菌中一种实现化合物多样性的方式。本论文以基因簇直接克隆与异源表达相结合的策略,成功实现了一个来源于纤维堆囊菌So0157-2中的NRPS-PKS杂合基因簇的异源表达,分离并鉴定了3个该基因簇对应的表达产物。本研究为后续从该菌株中挖掘更多活性天然产物奠定了技术基础,也为其他难培养菌株的次级代谢产物的挖掘提供了思路。
中图分类号:
周海波, 申琪瑶, 陈汉娜, 王宗杰, 李越中, 张友明, 卞小莹. 利用异源表达挖掘纤维堆囊菌So0157-2的新型天然产物[J]. 合成生物学, 2021, 2(5): 837-849.
Haibo ZHOU, Qiyao SHEN, Hanna CHEN, Zongjie WANG, Yuezhong LI, Youming ZHANG, Xiaoying BIAN. Genome mining for novel natural products in Sorangium cellulosum So0157-2 by heterologous expression[J]. Synthetic Biology Journal, 2021, 2(5): 837-849.
Strains/Plasmids | Description | Sources |
---|---|---|
Strains | [ | |
Escherichia coli GB05-dir | GB2005, araC-PBAD-ETgA; recE, recT, redγ, and recA regulated by arabinose-induced promoter are inserted at ybcC locus | |
E. coli GB05-red | GB2005, araC-PBAD-αβγA; redα, redβ, redγ, and recA regulated by arabinose-induced promoter are integrated at ybcC locus | [ |
S. cellulosum So0157-2 | wild type harboring BGC18 | [ |
S. brevitalea DSM 7029 | Burkholderiales strain DSM 7029, [Polyangium] brachysporum DSM 7029 (K481-B101; ATCC 53080) | [ |
DSM7029:Pkm-BGC18 | Pkm-BGC18 was inserted into genome of Schlegelella brevitalea DSM 7029, kmR | This work |
Plasmids | ||
p15A-cm-tetR-tetO-hyg-ccdB | direct cloning vector, p15A replicon, containing a tetracycline inducible promoter PtetO, cmR/hygR | [ |
pR6K-oriT-TnpA-IR-km | R6K replicon, containing MycoMar transposase gene (tnpA) and conjugation element oriT, kmR | [ |
p15A-cm-BGC18 | BGC18 was cloned into cloning vector, cmR | This work |
p15A-oriT-IR-Pkm-BGC18 | Expression vector of BGC18 with Pkm was inserted into upstream of the first core gene of BGC18, containing transposon elements oriT-tnpA-IR, kmR | This work |
表1 本研究所用的菌株和质粒
Tab. 1 Strains and plasmids used in this study
Strains/Plasmids | Description | Sources |
---|---|---|
Strains | [ | |
Escherichia coli GB05-dir | GB2005, araC-PBAD-ETgA; recE, recT, redγ, and recA regulated by arabinose-induced promoter are inserted at ybcC locus | |
E. coli GB05-red | GB2005, araC-PBAD-αβγA; redα, redβ, redγ, and recA regulated by arabinose-induced promoter are integrated at ybcC locus | [ |
S. cellulosum So0157-2 | wild type harboring BGC18 | [ |
S. brevitalea DSM 7029 | Burkholderiales strain DSM 7029, [Polyangium] brachysporum DSM 7029 (K481-B101; ATCC 53080) | [ |
DSM7029:Pkm-BGC18 | Pkm-BGC18 was inserted into genome of Schlegelella brevitalea DSM 7029, kmR | This work |
Plasmids | ||
p15A-cm-tetR-tetO-hyg-ccdB | direct cloning vector, p15A replicon, containing a tetracycline inducible promoter PtetO, cmR/hygR | [ |
pR6K-oriT-TnpA-IR-km | R6K replicon, containing MycoMar transposase gene (tnpA) and conjugation element oriT, kmR | [ |
p15A-cm-BGC18 | BGC18 was cloned into cloning vector, cmR | This work |
p15A-oriT-IR-Pkm-BGC18 | Expression vector of BGC18 with Pkm was inserted into upstream of the first core gene of BGC18, containing transposon elements oriT-tnpA-IR, kmR | This work |
Primers | Sequences (5′-3′) |
---|---|
BGC18-HAF | cagtatggccgatcggggtgtcagcggtcaacacgcgagctcgctcgctctgtggcggcgctctcatggtgcAACGCTCTCTACTAGAGTCA |
BGC18-HAR | gacgtgaccgggtatcgctaggtccacccaccaggagtccggccagggatccggagagtgagagttcaacgcGGGTCTTAAGACGTCGATATCT |
IR-Pkm-C18-HAF1 | gcctgcgatcgtaccattacgtatttttgcgcgagcggaacggtatgcagGCTGATCTTCAGATCCTCTAC |
IR-Pkm-C18-HAR1 | tcgagccgcaaggcgcattcttgctccagagagagcggcatagtacccaacctcctTCAGAAGAACTCGTCAAGAAG |
detect-C18 in 7029-F1 | GATGGGTTATCAGGACTACGC |
detect-C18 in 7029-R1 | CGAGGAGCCTGTAGAACGCGT |
detect-C18 in 7029-F2 | TCGACGATCAGGTGAAGATCCA |
detect-C18 in 7029-R2 | AGCTCTCCATAGGTGAGCGAA |
detect-C18 in 7029-F3 | TGAAGATCCGCGGCTATCGCA |
detect-C18 in 7029-R3 | CGGGAACTGGAACAGGTCCAT |
detect-C18 in 7029-F4 | TTCTTCGTCAACGCCGCGCC |
detect-C18 in 7029-R4 | GCGATAGGCGTGCACCGTGC |
detect-C18 in 7029-F5 | TCACGCTGCCGCAAGCACTC |
detect-C18 in 7029-R5 | GCTCTGCGAAGGACGTCCTC |
表2 本研究所用的引物
Tab. 2 Primers used in this study
Primers | Sequences (5′-3′) |
---|---|
BGC18-HAF | cagtatggccgatcggggtgtcagcggtcaacacgcgagctcgctcgctctgtggcggcgctctcatggtgcAACGCTCTCTACTAGAGTCA |
BGC18-HAR | gacgtgaccgggtatcgctaggtccacccaccaggagtccggccagggatccggagagtgagagttcaacgcGGGTCTTAAGACGTCGATATCT |
IR-Pkm-C18-HAF1 | gcctgcgatcgtaccattacgtatttttgcgcgagcggaacggtatgcagGCTGATCTTCAGATCCTCTAC |
IR-Pkm-C18-HAR1 | tcgagccgcaaggcgcattcttgctccagagagagcggcatagtacccaacctcctTCAGAAGAACTCGTCAAGAAG |
detect-C18 in 7029-F1 | GATGGGTTATCAGGACTACGC |
detect-C18 in 7029-R1 | CGAGGAGCCTGTAGAACGCGT |
detect-C18 in 7029-F2 | TCGACGATCAGGTGAAGATCCA |
detect-C18 in 7029-R2 | AGCTCTCCATAGGTGAGCGAA |
detect-C18 in 7029-F3 | TGAAGATCCGCGGCTATCGCA |
detect-C18 in 7029-R3 | CGGGAACTGGAACAGGTCCAT |
detect-C18 in 7029-F4 | TTCTTCGTCAACGCCGCGCC |
detect-C18 in 7029-R4 | GCGATAGGCGTGCACCGTGC |
detect-C18 in 7029-F5 | TCACGCTGCCGCAAGCACTC |
detect-C18 in 7029-R5 | GCTCTGCGAAGGACGTCCTC |
Gene Clusters | Type | Similar known cluster | Similarity |
---|---|---|---|
BGC1 BGC2 BGC3 BGC4 BGC5 BGC6 BGC7 BGC8 BGC9 BGC10 BGC11 BGC12 BGC13 BGC14 BGC15 BGC16 BGC17 BGC18 BGC19 BGC20 BGC21 BGC22 BGC23 BGC24 BGC25 BGC26 BGC27 BGC28 BGC29 BGC30 BGC31 BGC32 BGC33 BGC34 BGC35 | RiPP-like NRPS Thioamitides NRPS-PKS NRPS-PKS hglE-KS RiPP-like RiPP-like Indole T3PKS NRPS-T1PKS Terpene T1PKS NRPS RiPP-like thioamitides NRPS NRPS-T1PKS NRPS-T1PKS hglE-KS NRPS-T1PKS NRPS-T1PKS arylpolyene LAP, RRE-containing RiPP-like RiPP-like T1PKS T1PKS microbiridin phosphonate NRPS RiPP-like NRPS-T1PKS terpene NRPS | — — — pyxipyrrolone A/B kirromycin — — — — alkylpyrone-407/393 disorazol A geosmin pallasoren — — — myxochelin A/B — Epothilone — — Hapalosin N-tetradecanoyl tyrosine — — — — — microviridin J — coelibactin — crochelin A eremophikene — | — — — 11% 5% — — — — 6% 23% 100% 50% — — — 50% — 100% — — 40% 6% — — — — — 66% — 45% — 26% 100% — |
表3 antiSMASH 预测So0157-2基因组编码的生物合成基因簇
Tab. 3 Biosynthetic gene clusters in the genome of so0157-2 predicted by antiSMASH
Gene Clusters | Type | Similar known cluster | Similarity |
---|---|---|---|
BGC1 BGC2 BGC3 BGC4 BGC5 BGC6 BGC7 BGC8 BGC9 BGC10 BGC11 BGC12 BGC13 BGC14 BGC15 BGC16 BGC17 BGC18 BGC19 BGC20 BGC21 BGC22 BGC23 BGC24 BGC25 BGC26 BGC27 BGC28 BGC29 BGC30 BGC31 BGC32 BGC33 BGC34 BGC35 | RiPP-like NRPS Thioamitides NRPS-PKS NRPS-PKS hglE-KS RiPP-like RiPP-like Indole T3PKS NRPS-T1PKS Terpene T1PKS NRPS RiPP-like thioamitides NRPS NRPS-T1PKS NRPS-T1PKS hglE-KS NRPS-T1PKS NRPS-T1PKS arylpolyene LAP, RRE-containing RiPP-like RiPP-like T1PKS T1PKS microbiridin phosphonate NRPS RiPP-like NRPS-T1PKS terpene NRPS | — — — pyxipyrrolone A/B kirromycin — — — — alkylpyrone-407/393 disorazol A geosmin pallasoren — — — myxochelin A/B — Epothilone — — Hapalosin N-tetradecanoyl tyrosine — — — — — microviridin J — coelibactin — crochelin A eremophikene — | — — — 11% 5% — — — — 6% 23% 100% 50% — — — 50% — 100% — — 40% 6% — — — — — 66% — 45% — 26% 100% — |
图3 重组质粒p15A-cm-BGC18(a)和p15A-OriT-IR-Pkm-BGC18(b)分别以MscⅠ和ApaLⅠ酶切鉴定(红色方框代表酶切条带正确)
Fig. 3 a: Restriction analysis of the recombinant plasmids p15A-cm-BGC18 by MscⅠ(a) and p15A-OriT-IR-Pkm-BGC18 by ApaLⅠ(b)(Red box indicates right recombinant plasmids)
图4 BGC18在S. brevitalea DSM 7029中的异源表达产物LC-MS分析(BPC+:m/z 200~300)
Fig. 4 LC-MS analysis for the heterologous products of BGC18 expressed in S. brevitalea DSM 7029
No | 1 | 2 | 3 | ||
---|---|---|---|---|---|
δC | δH (J in Hz) | δH (J in Hz) | δH (J in Hz) | ||
N-Me-Leu | 1 2 3 4 5 6 N-Me | 167.6, C 59.2, CH 42.7, CH2 25.1, CH 23.1, CH3 21.8, CH3 32.1, CH3 | 3.78, dd (4.5, 8.2) 1.58, m 1.87, m 0.89, d (6.7) 0.93, d (6.7) 2.82, s | 3.79, t (6.7) 1.59, t (7.0) 1.79, m 0.90, d (6.5) 0.93, d (6.5) 2.82, s | 3.78, dd (4.5, 8.1) 1.58, m 1.87, m 0.89, d (6.5) 0.93, d (6.5) 2.82, s |
Val/Leu/Ile | 1 2 3 4 5 6 NH | 165.1, C 60.1, CH 32.6, CH 19.2, CH3 18.1, CH3 | 3.55, dd (3.6, 5.2) 2.01, m 0.92, d (6.8) 0.84, d (6.8) 8.24, d (2.4) | 3.73, dt (3.9, 8.7) 1.52, m 1.45, m 1.79, m 0.86, d (6.5) 0.90, d (6.5) 8.39, d (2.6) | 3.61, dd (3.8, 4.8) 1.73, m 1.43, m 1.11, m 0.84, t (6.5) 0.89, d (6.5) 8.23, d (1.6) |
表4 化合物1~3的NMR核磁数据
Tab. 4 1H (500 MHz) and 13C (125 MHz) data of 1~3 in DMSO-d6
No | 1 | 2 | 3 | ||
---|---|---|---|---|---|
δC | δH (J in Hz) | δH (J in Hz) | δH (J in Hz) | ||
N-Me-Leu | 1 2 3 4 5 6 N-Me | 167.6, C 59.2, CH 42.7, CH2 25.1, CH 23.1, CH3 21.8, CH3 32.1, CH3 | 3.78, dd (4.5, 8.2) 1.58, m 1.87, m 0.89, d (6.7) 0.93, d (6.7) 2.82, s | 3.79, t (6.7) 1.59, t (7.0) 1.79, m 0.90, d (6.5) 0.93, d (6.5) 2.82, s | 3.78, dd (4.5, 8.1) 1.58, m 1.87, m 0.89, d (6.5) 0.93, d (6.5) 2.82, s |
Val/Leu/Ile | 1 2 3 4 5 6 NH | 165.1, C 60.1, CH 32.6, CH 19.2, CH3 18.1, CH3 | 3.55, dd (3.6, 5.2) 2.01, m 0.92, d (6.8) 0.84, d (6.8) 8.24, d (2.4) | 3.73, dt (3.9, 8.7) 1.52, m 1.45, m 1.79, m 0.86, d (6.5) 0.90, d (6.5) 8.39, d (2.6) | 3.61, dd (3.8, 4.8) 1.73, m 1.43, m 1.11, m 0.84, t (6.5) 0.89, d (6.5) 8.23, d (1.6) |
Amino acid | Configuration | Retention time | |||
---|---|---|---|---|---|
Standard | 1 | 2 | 3 | ||
N-Me-L-Leu | L | 12.9 | 12.9 | 12.9 | 12.9 |
D | 13.2 | ||||
Val | L | 11.6 | 11.6 | ||
D | 12.5 | ||||
Leu | L | 29.7 | 29.7 | ||
D | 33.1 | ||||
Ile | L | 29.1 | 29.1 | ||
D | 32.5 | ||||
allo-Ile | L | 29.2 | |||
D | 32.6 |
表5 化合物1~3氨基酸与marfey试剂衍生产物的保留时间
Tab. 5 Retention time of amino acids derivatized with Marfey′s reagent
Amino acid | Configuration | Retention time | |||
---|---|---|---|---|---|
Standard | 1 | 2 | 3 | ||
N-Me-L-Leu | L | 12.9 | 12.9 | 12.9 | 12.9 |
D | 13.2 | ||||
Val | L | 11.6 | 11.6 | ||
D | 12.5 | ||||
Leu | L | 29.7 | 29.7 | ||
D | 33.1 | ||||
Ile | L | 29.1 | 29.1 | ||
D | 32.5 | ||||
allo-Ile | L | 29.2 | |||
D | 32.6 |
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