Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain
Abstract
:1. Introduction
2. Results
2.1. Determining of Nitrogen (N) Source and Doses of PAs for Synthetic (SN) Medium
2.1.1. Determining N Source for Cultivation of A. chrysogenum HY
2.1.2. Determining Doses of PAs for Cultivation of A. chrysogenum HY on SN Medium with L-Asn
2.2. Submerged Fermentation of A. chrysogenum HY Strain with Exogenous PAs on SN and CP Media
2.3. Effect of the Addition of Exogenous PAs to SN and CP Media on Cephems Ratio
2.4. Analysis of the Expression Level of the Biosynthetic and Transport Genes from Beta-Lactam BGCs in A. chrysogenum HY after Fermentation on SN and CP Media with PAs
2.5. Effect of the Addition of Exogenous PAs to SN and CP Media on Acetyl-CoA Content
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Strains of Microorganisms
4.3. Cultivation of A. chrysogenum Strains on Agarized Media with PAs
4.4. Light Microscopy
4.5. Submerged Fermentation of A. chrysogenum HY Strain with Exogenous PAs
4.6. Determination of Dry Biomass
4.7. HPLC Analysis of Beta-Lactams
4.8. Determination of Cellular Acetyl-CoA
4.9. Preparation of Total RNA and cDNA Synthesis and qPCR Analysis
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
Acetyl-CoA | Acetyl-coenzyme A |
BGC | biosynthetic gene cluster |
CAA | Agarized Czapek-L-Asn (medium) |
CPC | Cephalosporin C |
CDA | Agarized Czapek-Dox (medium) |
CPA | Agarized complex (medium) |
DAOC | Deacetoxycephalosporin C |
1,3-DAP | 1,3-diaminopropane |
DAC | Deacetylcephalosporin C |
LLD-ACV | δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine |
HY | High-yielding |
PAs | Polyamines |
SN | Synthetic (medium) |
CP | Complex (medium) |
SPD | Spermidine |
WT | Wild type |
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Primer | Gene | Product, Function | Oligonucleotide (Sequence 5 → 3) | GenBank, Source |
---|---|---|---|---|
actq1 | act1 | γ-Actin, a major component of the cytoskeleton | CCGGTTTCGCCGGTGATGATGCT | JN836733.1, [50] |
actq2 | TGCTCAATGGGGTAGCGCAG | |||
pcbABq3 | pcbAB | δ-(L-α-Aminoadipyl)-L-Cysteinyl-D-Valine synthetase | AGGCATCGTCAGGTTGGCCG | E05192.1, [36] |
pcbABq4 | CCGGAGGGGCCATACCACAT | |||
pcbCq1 | pcbC | Isopenicillin N-synthase | CTAGGTCGCGACGAGGACTTCT | M33522.1, [36] |
pcbCq2 | CACGTCGGACTGGTACAACACC | |||
cefD1q1 | cefD1 | Isopenicillin N-CoA synthetase | CCCCGGTGAGGAAGATGCGT | AJ507632.2, [36] |
cefD1q2 | TCGATCTCCGCCTTGGACGC | |||
cefD2q1 | cefD2 | Isopenicillin N-CoA epimerase | ACAGGATGGAGAGGAGCACCTTG | |
cefD2q2 | TCGTAGAGCTCGCGGGGCTA | |||
cefEFq3 | cefEF | Deacetoxycephalosporin C synthetase/ hydroxylase | GTCGAGTGCGATCCCCTCCT | AJ404737.1, [40] |
cefEFq4 | CGAATTCTCCGTCCACCTCG | |||
cefGq3 | cefG | Deacetylcephalosporin-C acetyltransferase | CTCCTGGAGCCATATGGAAGCGC | M91649.1, [40] |
cefGq4 | GGTGCGCAGCTTGGTTCGAGAC | |||
cefPq1 | cefP | Multidrug efflux pump protein, putative role in isopenicillin N transport | AATGCGACCCCGAGGAGTACGT | AM231816.1, [36] |
cefPq2 | CCATCCCAGGAATGTTGTCGGC | |||
cefMq1 | cefM | Multidrug efflux pump protein, putative role in penicillin N transport | TTTATCCAGGAGGAGCGCGGTC | AM231815.1, [36] |
cefMq2 | TGTCGTAGGCGGTTCACCTTGC | |||
cefTq3 | cefT | Multidrug efflux pump protein | TGTTGTCGGATTCGGTGTCGG | AJ487683.1, [50] |
cefTq4 | TTCCACATATCGGCAAGGGTGC |
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Zhgun, A.A.; Eldarov, M.A. Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain. Int. J. Mol. Sci. 2022, 23, 14625. https://doi.org/10.3390/ijms232314625
Zhgun AA, Eldarov MA. Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain. International Journal of Molecular Sciences. 2022; 23(23):14625. https://doi.org/10.3390/ijms232314625
Chicago/Turabian StyleZhgun, Alexander A., and Mikhail A. Eldarov. 2022. "Spermidine and 1,3-Diaminopropane Have Opposite Effects on the Final Stage of Cephalosporin C Biosynthesis in High-Yielding Acremonium chrysogenum Strain" International Journal of Molecular Sciences 23, no. 23: 14625. https://doi.org/10.3390/ijms232314625