p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation
Abstract
:1. Introduction
2. Results
2.1. Genome Sequencing Revealed a Circular Chromosome of Strain DSM 44131T
2.2. Comparative Genomic Analysis within Actinosynnema Resulted in the Discovery of Dnacin B1 BGC
2.3. Functional Genes Involved in the Assembly of Dnacin B1 Skeleton
2.4. Reconstitution of PAPA Pathway Revealed TyrB Participating in the Mature of PAPA
2.5. Aminotransferases Responsible for the PAPA Maturation Dispersed within the Genome
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Plasmids and Reagents
4.2. General Experimental Procedures
4.3. DNA Sequencing for Strain DSM 44131T
4.4. Bioinformatics Analysis
4.5. Construction of the Dnacin B1 BGC Inactivation Mutant
4.6. Optimization of Fermentation Medium
4.7. Total RNA Extraction and q-PCR Analysis
4.8. Production and Detection of Dnacin B1 in Strain DSM 44131T
4.9. Reconstruction of PAPA Biosynthesis Pathway in E. coli
4.10. Protein Expression and Purification
4.11. Enzyme Activity Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are not available from the authors. |
ORF | Number of Amino Acids | Proposed Function | Sequence Similarity (Protein, Origin) | % Identity/ Similarity | Accession No. |
---|---|---|---|---|---|
DinR1 | 951 | ABC transporter | IQ63_41860, Streptomyces acidiscabies | 64/75 | KND24720.1 |
ORF1 | 117 | unknown | SD37_10880, Amycolatopsis orientalis | 55/75 | ANN21703.1 |
ORF2 | 175 | protease inhibitor | AC230_04780, Streptomyces caatingaensis | 38/54 | KNB54254.1 |
ORF3 | 395 | histidine kinase | A9W97_15640, Mycobacterium gordonae | 51/60 | OBJ88517.1 |
ORF4 | 159 | ATP-binding protein | RKT69099.1, Saccharothrix variisporea | 45/51 | DFJ66_2293 |
ORF5 | 306 | epimerase | DI639_00155, Leifsonia xyli | 74/83 | PZO61411.1 |
DinR2 | 228 | TetR family transcriptional regulator | STRAU_2618, Streptomyces aurantiacus | 58/71 | EPH44178.1 |
DinA | 534 | flavin adenine dinucleotide (FAD)-linked oxidase | NapU, Streptomyces lusitanus | 64/75 | AGD80628.1 |
DinB | 471 | gamma-aminobutyraldehyde dehydrogenase | MCBG_02966, Micromonospora sp. M42 | 53/65 | EWM65833.1 |
DinC | 741 | peptidase | NapG, Streptomyces lusitanus | 48/56 | AGD80614.1 |
DinD | 183 | flavin mononucleotide (FMN) reductase | SsuE, Streptomyces noursei | 58/67 | WP_067345371.1 |
DinR3 | 707 | regulatory protein | NapR3, Streptomyces lusitanus | 55/68 | AGD80615.1 |
DinE | 103 | 4-amino-4-deoxychorismate mutase | PapB, Streptomyces venezuelae | 34/53 | BAD21142.1 |
DinF | 307 | 4-amino-4-deoxyprephenate dehydrogenase | PapC, Streptomyces venezuelae | 40/51 | BAD21141.1 |
DinG | 300 | unknown | CLV43_102760, Umezawaea tangerina | 47/57 | PRY45195.1 |
DinH | 796 | adenylation domain | NapH, Streptomyces lusitanus | 51/60 | AGD80616.1 |
DinI | 342 | non-heme iron hydroxylase | NapI, Streptomyces lusitanus | 63/75 | AGD80617.1 |
DinJ | 1482 | NRPS | NapJ, Streptomyces lusitanus | 68/79 | AGD80618.1 |
Module 5 | C-A-PCP-RE | ||||
DinK | 66 | MbtH-like protein | NapK, Streptomyces lusitanus | 73/84 | AGD80619.1 |
DinL | 1125 | NRPS | NapL, Streptomyces lusitanus | 54/64 | AGD80620.1 |
Module 4 | C-A-PCP | ||||
DinM | 252 | thioesterase II | NapM, Streptomyces lusitanus | 51/64 | AGD80621.1 |
DinN | 632 | AMP-dependent synthetase and ligase | NapN, Streptomyces lusitanus | 64/74 | AGD80622.1 |
Loading | AL-PCP | ||||
DinO | 3049 | NRPS | NapO, Streptomyces lusitanus | 52/63 | AGD80623.1 |
Module 1 | C-A-PCP | ||||
Module 2 | C-A-PCP | ||||
Module 3 | C-A-PCP | ||||
DinP | 223 | hypothetical protein | NapP, Streptomyces lusitanus | 45/59 | AGD80633.1 |
DinR4 | 909 | ABC transporter | B0I31_10111, Saccharothrix carnea | 56/67 | PSL57800.1 |
DinR5 | 280 | ABC transporter | CLV69_102810, Yuhushiella deserti | 53/69 | TDX97704.1 |
DinQ | 336 | 3-oxoacyl-synthase III (KS) | NapE, Streptomyces lusitanus | 72/80 | AGD80612.1 |
DinS | 754 | Transketolase | NapD, Streptomyces lusitanus | 59/68 | AGD80611.1 |
DinT | 76 | acyl carrier protein (ACP) | NapC, Streptomyces lusitanus | 63/75 | AGD80633.1 |
DinU | 306 | transketolase | NapB, Streptomyces lusitanus | 68/77 | AGD80609.1 |
DinV | 706 | 4-amino-4-deoxychorismate synthase | PapA, Streptomyces venezuelae | 48/58 | BAD21140.1 |
DinW | 486 | 4-hydroxyphenylacetate 3-monooxygenase | EWI31_24745, Streptomyces tsukubensis | 56/69 | TAI42134.1 |
DinR6 | 613 | ABC transporter | CLV43_102777, Umezawaea tangerina | 70/78 | PRY45212.1 |
DinR7 | 601 | ABC transporter | DIU55_13605, Firmicutes bacterium | 51/66 | PZN68908.1 |
DinX | 477 | monooxygenase | NapA, Streptomyces lusitanus | 69/76 | AGD80608.1 |
DinR8 | 818 | UV-repair protein | NapR1, Streptomyces lusitanus | 75/84 | AGD80606.1 |
DinY | 244 | methyltransferase | QncJ, Streptomyces melanovinaceus | 47/65 | AGD95052.1 |
DinZ | 118 | long-chain-fatty-acid--CoA ligase | NCTC13184_06982, Nocardia africana | 60/81 | SUA48430.1 |
ORF6 | 182 | sugar O-acetyltransferase | E1091_12580, Micromonospora fluostatini | 64/72 | TDB92781.1 |
DinR9 | 304 | LysR family transcriptional regulator | AFR_16085, Actinoplanes friuliensis | 57/69 | AGZ41499.1 |
ORF7 | 104 | transposase | CLV43_10550, Umezawaea tangerina | 56/61 | PRY41292.1 |
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Hu, X.; Li, X.; Sheng, Y.; Wang, H.; Li, X.; Ou, Y.; Deng, Z.; Bai, L.; Kang, Q. p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation. Molecules 2020, 25, 4186. https://doi.org/10.3390/molecules25184186
Hu X, Li X, Sheng Y, Wang H, Li X, Ou Y, Deng Z, Bai L, Kang Q. p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation. Molecules. 2020; 25(18):4186. https://doi.org/10.3390/molecules25184186
Chicago/Turabian StyleHu, Xiaojing, Xing Li, Yong Sheng, Hengyu Wang, Xiaobin Li, Yixin Ou, Zixin Deng, Linquan Bai, and Qianjin Kang. 2020. "p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation" Molecules 25, no. 18: 4186. https://doi.org/10.3390/molecules25184186