Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cloning and Sequencing of the spc Gene Cluster from S. sanyensis FMA
2.2. Organization and Characterization of the spc Gene Cluster
Protein | Size (aa) | Proposed function | Homolog in strain TP-A0274 (Accession No.) | % Identity/Similarity |
---|---|---|---|---|
Orf(-1) | 341 | hypothetical protein | - | - |
SpcR | 986 | transcriptional regulator | StaR (BAC55205.1) | 62/70% |
SpcB | 357 | dTDP-glucose 4,6-dehydratase | StaB (BAC55206.1) | 82/87% |
SpcA | 354 | glucose-1-phosphate thymidyltransferase | StaA (BAC55207.1) | 78/87% |
SpcN | 390 | cytochrome P450 | StaN (BAC55208.1) | 76/81% |
SpcG | 433 | N-glycosyltransferase | StaG (BAC55209.1) | 76/84% |
SpcO | 503 | L-amino acid oxidase | StaO (BAC55210.1) | 77/85% |
SpcD | 1123 | chromopyrrolic acid synthase | StaD (BAC55211.1) | 65/71% |
SpcP | 427 | cytochrome P450 | StaP (BAC55212.1) | 71/79% |
SpcMA | 277 | methyltransferase | StaMA (BAC55213.1) | 63/72% |
SpcJ | 477 | 2,3-dehydratase | StaJ (BAC55214.1) | 64/70% |
SpcK | 331 | 4-ketoreductase | StaK (BAC55215.1) | 75/83% |
SpcI | 369 | aminotransferase | StaI (BAC55216.1) | 86/92% |
SpcE | 208 | 3,5-epimerase | StaE (BAC55217.1) | 83/89% |
SpcMB | 281 | methyltransferase | StaMB (BAC55218.1) | 75/83% |
SpcC | 542 | monooxygenase | StaC (BAF47693.1) | 78/86% |
Orf1 | 238 | hypothetical protein | - | - |
Orf2 | 540 | integral membrane protein | - | - |
Orf3 | 292 | outer membrane adhesion like protein | - | - |
Orf4 | 660 | integral membrane protein | - | - |
Orf5 | 884 | D-alanyl-D-alanine carboxypeptidase | - | - |
2.3. Involvement of the spc Gene Cluster in ICZs Biosynthesis in S. sanyensis FMA
2.4. Heterologous Expression of the spc Gene Cluster in Streptomyces coelicolor M1152
3. Experimental Section
3.1. Bacterial Strains, Plasmids and Reagents
3.2. DNA Manipulation, Sequencing and Bioinformatic Analysis
3.3. Genomic Library Construction
3.4. Library Screening
3.5. Gene Inactivation
3.6. Heterologous Expression of the spc Gene Cluster in S. coelicolor M1152
3.7. Production and Analyses of ICZs in S. sanyensis FMA Strains
3.8. Nucleotide Sequence Accession Number
4. Conclusions
Supplementary Material
Strains or plasmids | Description | Reference or source |
---|---|---|
Strains | ||
E. coli Top10 | Host strain of cosmid vector SuperCos1 | Invitrogen |
E. coli DH5a | Host strain for general cloning | Stratagene |
E. coli ET12567/pUZ8002 | Host strain for conjugation | [39] |
E. coli BW25113/pIJ790 | Host strain for PCR-targeting | [40] |
Strptomyces sanyensis FMA | Wild type, ICZs producer | [4] |
LIW601 | Δ spcC inactivation mutant of S. sanyensis FMA | This study |
LIW602 | Δ spcI inactivation mutant of S. sanyensis FMA | This study |
LIW603 | Δ spcR inactivation mutant of S. sanyensis FMA | This study |
Strptomyces coelicolor M1152/pWLI617 | Strptomyces coelicolor M1152 carrying pWLI617 | This study |
Plasmids | ||
SuperCosI | Apr, Kmr, cosmid vector | Stratagene |
pUM-T | Apr, general cloning vector | Bioteke |
pIJ773 | Aprr, source of acc(3)IV-oriT cassette | [34] |
pIJ790 | Cmr, λ RED recombination plasmid | [34] |
pWLI601 | pUM-T cloned with the amplified FAD-dependent monooxygenase gene fragment | This study |
pWLI611-615 | Genomic library cosmids harboring spc biosynthetic genes from S. sanyensis FMA | This study |
pWLI621 | pWLI615 derivative where spcC was replaced with acc(3)IV-oriT cassette | This study |
pWLI622 | pWLI615 derivative where spcI was replaced with acc(3)IV-oriT cassette | This study |
pWLI623 | pWLI615 derivative where spcR was replaced with acc(3)IV-oriT cassette | This study |
pWLI617 | pWLI615 derivative which was equipped with oriT and φC31 attP/int | This study |
pSET152AB | pSET152 derivative | [41] |
Gene | Primer pairs used for inactivation (5'→3') |
---|---|
spcC | spcCMF: GCCGCCCGACATCCTGGTGGACGGGCCCGAGGGCGACCGGattccggggatccgtcgacc |
spcCMR: CGCTCGTACAGCTTGGCGACCTCCTCGCCGCCCCCGCCGCGtgtaggctggagctgcttc | |
spcI | spcIMF: CATGACCACGCGAGTATGGGACTACCTGGCGGAGTACGAGattccggggatccgtcgacc |
spcIMR: CCTCACAGCGTCTCCAGCACCTCGCGCAGCGCGTGGACGACtgtaggctggagctgcttc | |
spcR | spcRMF: CATGGGTCCTCAGGTACGAGCGTTAGCACCGCTGCGCGGCattccggggatccgtcgacc |
spcRMR: CCTCAGGTCCGGAAGCGCACCAGAGCGGTGCGGGAGCGTATtgtaggctggagctgcttc |
Gene | Primer pairs designed to verify the mutant strains (5'→3') | Length of fragment replaced | Length of desired PCR fragments | |
---|---|---|---|---|
Wild type | Mutant | |||
spcC | spcCCF: AGTTGCCGCCCGACATCC | 321 bp | 409 bp | 1470 bp |
spcCCR: GCCCGCTCGTACAGCTTGG | ||||
spcI | spcICF: GACCACGCGAGTATGGGACTAC | 1032 bp | 1111 bp | 1467 bp |
spcICR: GCCTCACAGCGTCTCCAGCA | ||||
spcR | spcRCF: GGTCCCGTCCCCTTCGACA | 2883 bp | 3001 bp | 1540 bp |
spcRCR: GGCCTCAGGTCCGGAAGC |
Position | δH (numbers of H, multiplicity b, J, Hz) |
---|---|
1 | 7.30 (1H, d, 7.9) |
2 | 7.48 (1H, dt, 7.7, 1.1) |
3 | 7.36 (1H, dt, 7.5, 0.5) |
4 | 9.41 (1H, d, 7.9) |
6 | 6.21 (1H, brs) |
7 | 5.03 (2H, AB, 16.1) |
8 | 7.90 (1H, d, 7.7) |
9 | 7.32 (1H, t, 7.5) |
10 | 7.42 (1H, dt, 7.7,1.1) |
11 | 7.93 (1H, d, 8.4) |
3′ | 3.88 (1H, d,3.6) |
4′ | 3.35 (1H, m) |
5′ | 2.41 (1H, ddd, 14.9,5.7,3.7), 2.75 (1H, ddd, 14.9,4.0,1.2) |
6′ | 6.57(1H, dd, 5.7,1.1) |
2′-CH3 | 2.36 (1H, s) |
3′-OCH3 | 3.41 (1H, s) |
4′-NCH3 | 1.54 (1H, s) |
Position | δH(multiplicity b, J, Hz) | δC |
---|---|---|
1 | 7.70 (d, 8.1) | 111.9 |
2 | 7.43 (dt, 7.7, 1.1) | 125.5 |
3 | 7.22 (dt, 7.5, 0.8) | 119.4 |
4 | 9.20(d, 7.9) | 125.3 |
6 | 8.44 (brs) | / |
7 | 4.96 (s) | 45.7 |
8 | 8.04 (d, 7.7) | 121.6 |
9 | 7.31 (dt, 7.5, 0.8) | 120.2 |
10 | 7.48 (dt, 7.7,1.1) | 125.3 |
11 | 7.77 (d, 8.1) | 112.2 |
12 | 11.70(brs) | / |
13 | 11.52(brs) | / |
Position | δH(multiplicity b, J, Hz) | δC |
---|---|---|
1 | 7.69 (d, 8.5) | 110.3 |
2 | 7.48 (m) | 125.4 |
3 | 7.27 (dt, 7.5, 0.8) | 119.7 |
4 | 9.45(d, 7.8) | 125.8 |
4a | / | 122.9 |
4c | / | 118.8 |
5 | / | 172.4 |
6 | 8.53 (brs) | / |
7 | 5.00 (AB,17.5) | 45.6 |
7a | / | 134.4 |
7b | / | 115.4 |
7c | / | 122.5 |
8 | 8.06 (d, 7.8) | 121.5 |
9 | 7.31 (dt, 7.4, 0.9) | 120.2 |
10 | 7.50 (m) | 125.4 |
11 | 7.60 (d, 8.1) | 111.8 |
11a | / | 139.4 |
12 | 11.68 (brs) | / |
12b | / | 124.9 |
13a | / | 140.5 |
1′ | 6.39 (d, 9.6) | 77.3 |
2′ | 4.48 (m) | 67.2 |
3′ | 4.17 (dd, 3.6, 5.9) | 72.1 |
4′ | 4.04 (dd, 1.0, 3.6) | 71.9 |
5′ | 4.47 (m) | 76.7 |
6′ | 1.69 (d, 7.3) | 15.8 |
Acknowledgments
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Li, T.; Du, Y.; Cui, Q.; Zhang, J.; Zhu, W.; Hong, K.; Li, W. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA. Mar. Drugs 2013, 11, 466-488. https://doi.org/10.3390/md11020466
Li T, Du Y, Cui Q, Zhang J, Zhu W, Hong K, Li W. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA. Marine Drugs. 2013; 11(2):466-488. https://doi.org/10.3390/md11020466
Chicago/Turabian StyleLi, Tong, Yuanyuan Du, Qiu Cui, Jingtao Zhang, Weiming Zhu, Kui Hong, and Wenli Li. 2013. "Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA" Marine Drugs 11, no. 2: 466-488. https://doi.org/10.3390/md11020466
APA StyleLi, T., Du, Y., Cui, Q., Zhang, J., Zhu, W., Hong, K., & Li, W. (2013). Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA. Marine Drugs, 11(2), 466-488. https://doi.org/10.3390/md11020466