Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum
Abstract
:1. Introduction
2. Results
2.1. Bioinformatic Identification of the Terpenoid Synthase Candidate Gene
2.2. Functional Testing of FPRN_05485
2.3. Functional Analysis of the FUP Gene Cluster
2.3.1. Preparation of Knock-Out Strains
2.3.2. Search for Predicted FUP Intermediates and Structurally Related Compounds
2.3.3. Abundance of FUP and Pathway Precursors in Knock-Out Strains
2.3.4. Structure Similarity Determination of Suspect Compounds with Identified Terpestacin and FUP via LC-HRMS/MS
3. Discussion
3.1. The Proposed Fusaproliferin Biosynthetic Pathway in F. proliferatum
3.2. Possible Reasons for the Inability to Obtain a FUP2 (FPRN_05484) Mutant
3.3. Significantly Higher Terpestacin in the fup5 Mutant Strain
3.4. Perspectives and Open Questions
4. Material and methods
4.1. Strains
4.2. Preparation of Knock-Out Strains
4.3. Toxin Test and Extraction of Secondary Metabolites
4.4. LC-HRMS/MS-Based Target Screening and Suspect Analysis of Intermediates of Fusaproliferin Biosynthetic Pathway
4.4.1. Chemicals and Standards
4.4.2. Purification Fusaproliferin and Terpestacin
4.4.3. LC-HRMS/MS Measurement
Liquid Chromatography
Mass Spectrometry
4.4.4. Feature-Based Molecular Networking
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Designation | Annotation | NRRL 62905 Gene Protein | ET1 Gene Protein | Protein Identity |
---|---|---|---|---|
Prolyl-tRNA synthetase | FPRN_05483 (CVK87167) * | FPRO_05644 CZR39164 | Diff. N-terminus 217/365 (59.5%) | |
FpFUP2 | Cytochrome P-450 monooxygenase (CYP65A1) | FPRN_05484 CVK87169 | FPRO_05645 CZR39163 | 506/509 (99.4%) |
FpFUP1 | Geranylgeranyl diphosphate synthase, TPS | FPRN_05485 CVK87172 | FPRO_05646 CZR39162 | 744/753 (98.8%) |
FpFUP4 | FAD linked oxidase, N-terminal | FPRN_05486 CVK87175 | FPRO_05647 CZR39161 | 506/519 (97.5%) |
FpFUP5 | Acyltransferase-related | FPRN_05487 CVK87178 | FPRO_05648 CZR39160 | 375/390 (96.2%) |
FpFUP3 | Cytochrome P450 | FPRN_05488 CVK87181 | FPRO_05649 CZR39159 | 521/529 (98.5%) |
(FpFUP6) | Transcription factor domain, fungi | FPRN_05489 CVK87185 | FPRO_05650 CZR39158 | 694/700 (99.1%) |
(FpFUP7) | Zn(II)2Cys6 transcription factor | FPRN_05490 CVK87187 | FPRO_05651 CZR39157 | 235/245 (95.9%) |
Lactate/Malate dehydrogenase | FPRN_05491 CVK87189 | PRO_05652 CZR39156 | 318/325 (97.8%) |
Fragment | Primer (5´–3´) | Length (bp) |
---|---|---|
FUP1-5´ | TAATGGCCGCATAGGCCGCGTCAACAGCTGCTCCC | 620 |
AAACTAGTCGGTTAGTCTTTGCGAGCTCTCC | ||
FUP1-3´ | AATGTCGACGCCTCATTTACACGGACTACAAGGTC | 593 |
ACAAGCTTGAAATGTGCAGCATGGCGGTG | ||
FUP2-5´ | TAATGGCCGCATAGGCCTTGCATTATCCTGTTACCTTACTC | 640 |
AAACTAGTCGGTTAGTCTTTGCGAGCTCTCC | ||
FUP2-3´ | AATGTCGACGCCTCATTTACACGGACTACAAGGTC | 576 |
ACAAGCTTGAAATGTGCAGCATGGCGGTG | ||
FUP3-5´ | TAATGGCCGCATAGGCCGCAGGTTTAGCTCTGCCTGTAGG | 732 |
AAACTAGTCAAGTCGTCTTGAGATGATGACAAGCTC | ||
FUP3-3´ | AATGTCGACGCTCTTTGAGACGACCGCTG | 599 |
ACAAGCTTCGAGGCAGAAGAGGGAGTAACC | ||
FUP4-5´ | TAATGGCCGCATAGGCCGTAAACGCCACCATGCTCAGC | 650 |
AAACTAGTGCGAAACCAAGGATTCTGAATGCCG | ||
FUP4-3´ | AATGTCGACGTTCACGACAGCCACCTTCAGG | 671 |
ACAAGCTTGACCGCATTCATAATTGGGCAA | ||
FUP5-5´ | TAATGGCCGCATAGGCCGCCGTTGAATTACCCAGATCCCA | 609 |
AAACTAGTGTTGAGAGTACAGTACAGGTGCAATG | ||
FUP5-3´ | AATGTCGACCCTGAGCAGATTCTGGAACCGC | 539 |
ACAAGCTTCCTCATATCCACAACAATACACTAGATTGCC |
Region | Outside (5’–3’) | Gene specific (5’–3’) |
---|---|---|
FUP1-5´ | GATGGGGCGTCAGTTCTTGAGATCG | CCGTCTCTTGAGCTTCCTCTGCC |
FUP1-3´ | CGCCCTATCAAGCCAGAGTGCC | GAGCACAGCTACGGAGACACCAG |
FUP4-5´ | GCATAACGACCTCTCTATAGG | GGAGCAAGGAGGACACTGG |
FUP4-3´ | CGCACCTGGACTTGTGACC | GTCACGTCACTATCGGACCTTG |
FUP5-5´ | CGTCGGCAACTGACCCTAACC | CAAGACCAGAATCGGAGTAGTGAGC |
FUP5-3´ | CAAGATCTAATGGTGATCTTC | GTCCACCCAGTAAACGGACCTTG |
FUP3-5´ | GCGTCTGACATGCCGAGAGATG | CCATCGAACGGAAATGGTATTGGCC |
FUP3-3´ | CGTTCTGGACTTTGCTGGGTAC | GGGTTTACGGGACGGAGGAAG |
Nr. | Molecular Formula | Monoisotopic Mass | Retention Time | Adduct Type | m/z | Compound Annotated/Identified |
---|---|---|---|---|---|---|
1 | C25H42O | 358.3236 | 34.28 | [M−H2O+H]+ | 341.3203 | Preterpestacin I |
[M+H]+ | 359.3308 | |||||
[M+Na]+ | 381.3128 | |||||
[M+NH4]+ | 376.3574 | |||||
2 | C25H42O2 | 374.3185 | 32.44 | [M−H2O+H]+ | 357.3152 | Preterpestacin II a |
[M+H]+ | 375.3258 | |||||
[M+Na]+ | 397.3077 | |||||
[M+NH4]+ | 392.3523 | |||||
[M+HCOO]− | 419.3167 | |||||
[M+Cl]− | 409.2868 | |||||
3 | C25H40O2 | 372.3028 | 33.05 | [M−H2O+H]+ | 355.2995 | Preterpestacin II c |
[M+H]+ | 373.3101 | |||||
[M+Na]+ | 395.2921 | |||||
[M+NH4]+ | 390.3367 | |||||
4 | C25H40O3 | 388.2977 | 32.15 | [M−H2O+H]+ | 371.2945 | Preterpestacin II b |
[M+Na]+ | 411.2870 | |||||
[M−H]− | 387.2905 | |||||
[M+HCOO]− | 433.2959 | |||||
[M+Cl]− | 423.2660 | |||||
5 | C25H42O4 | 406.3083 | 27.02 | [M−H2O+H]+ | 389.3050 | Preterpestacin III |
[M+Na]+ | 429.2975 | |||||
[M+HCOO]− | 451.3065 | |||||
6 | C25H40O4 | 404.2927 | 26.4 | [M−H2O+H]+ | 387.2894 | Two tautomers keto or enol |
[M+Na]+ | 427.2819 | |||||
[M−H]− | 403.2854 | |||||
[M+HCOO]− | 449.2909 | |||||
7 | C25H38O4 | 402.2770 | 27.73 | [M−H2O+H]+ | 385.2737 | Terpestacin keto from |
[M+Na]+ | 425.2662 | |||||
[M−H]- | 401.2697 | |||||
[M+HCOO]− | 447.2752 | |||||
[M+Cl]− | 437.2453 | |||||
8 | C25H38O4 | 402.2770 | 26.6 | [M−H2O+H]+ | 385.2737 | Terpestacin enol form TPC |
[M+H]+ | 403.2843 | |||||
[M+Na]+ | 425.2662 | |||||
[M−H]− | 401.2697 | |||||
[M+HCOO]- | 447.2752 | |||||
[M+Cl]− | 437.2453 | |||||
9 | C25H36O4 | 400.2614 | 26.87 | [M−H2O+H]+ | 383.2581 | Terpestacin aldehyde |
[M+H]+ | 401.2686 | |||||
[M−H]− | 399.2541 | |||||
10 | C27H40O5 | 444.2876 | 27.57 | [M−H2O+H]+ | 427.2843 | Fusaproliferin FUP |
[M+H]+ | 445.2949 | |||||
[M+Na]+ | 467.2768 | |||||
[M+NH4]+ | 462.3214 | |||||
[M−H]− | 443.2803 | |||||
11 | C25H40O | 356.3079 | 34.2 | [M−H2O+H]+ | 339.3046 | Oxo-preterpestacin I |
[M+H]+ | 357.3152 | |||||
[M+Na]+ | 379.2971 |
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Ćeranić, A.; Svoboda, T.; Berthiller, F.; Sulyok, M.; Samson, J.M.; Güldener, U.; Schuhmacher, R.; Adam, G. Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum. Toxins 2021, 13, 468. https://doi.org/10.3390/toxins13070468
Ćeranić A, Svoboda T, Berthiller F, Sulyok M, Samson JM, Güldener U, Schuhmacher R, Adam G. Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum. Toxins. 2021; 13(7):468. https://doi.org/10.3390/toxins13070468
Chicago/Turabian StyleĆeranić, Asja, Thomas Svoboda, Franz Berthiller, Michael Sulyok, Jonathan Matthew Samson, Ulrich Güldener, Rainer Schuhmacher, and Gerhard Adam. 2021. "Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum" Toxins 13, no. 7: 468. https://doi.org/10.3390/toxins13070468
APA StyleĆeranić, A., Svoboda, T., Berthiller, F., Sulyok, M., Samson, J. M., Güldener, U., Schuhmacher, R., & Adam, G. (2021). Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum. Toxins, 13(7), 468. https://doi.org/10.3390/toxins13070468