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| Status |
Public on Aug 26, 2014 |
| Title |
Comparative genomics reveals new molecular targets for accelerated strain improvement of rifamycin B-producing strains |
| Organism |
Amycolatopsis mediterranei S699 |
| Experiment type |
Expression profiling by array
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| Summary |
In the present study, we have used a genomic approach to understand how the classical mutate-and-screen method actually generated an improved rifamycin B producer. Compared with the reference strains Amycolatopsis mediterranei S699 (rifamycin B producer) and U32 (rifamycin SV producer), a total of 250 variations affecting a total of 227 coding sequences (CDS) were found in rifamycin B overproducing strain HP-130. One hundred nine CDS variations were specific to HP-130 as they were absent in both S699 and U32. A lot of variations affected genes coding for fatty acid (an lipid) metabolism, which is tightly interconnected with rifamycin biosynthesis by common metabolic precursors (malonyl-CoA, methylmalonyl-CoA). Interestingly, a nonsense mutation was mapped within mutB coding for methylmalonyl-CoA mutase suggesting the importance of metabolic re-direction of carbon flow toward rifamycin biosynthesis in the over-producing phenotype of HP-130. Other key mutations were: i.) a missense mutation affecting ppk, which encodes a polyphosphate kinase and was previously shown to play a negative role in the control of antibiotic biosynthesis in Streptomyces lividans; ii.) a missense mutation in argS2 affecting one of the two arginyl tRNA synthetases of A. mediterranei; iii.) a missense mutation in rifN coding for a protein belonging to the ROK family of transcriptional regulators with kanosamine kinase activity. Microarray analysis of the transcriptome of HP-130 as compared to that of S699 was useful to understand the effects of several mutations on global gene expression profile. Genomic and transcriptomic data were used to improve rifamycin B production in S699 by genetic engineering thus proving the causative effect of the above-mentioned mutations on the overproducing phenotype.
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| Overall design |
In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of a rifamycin B overproducer (HP-130), which was obtained by the traditional mutate-and-screen method. To gain insight about the mechanisms underlying improved rifamycin B production in HP-130, DNA microarray of A. mediterranei were manufactured and used for comparative analysis of the gene expression profile in HP-130 and S699. In DNA microarray experiments the two strains were cultivated under standard batch-culture conditions in modified RFB 2244 fermentation medium, and samples were collected at different time points (24-72 h) for RNA extraction. Gene expression data were analyzed to identify transcripts modulated during the growth curve. Amycolatopsis mediterranei S699 (WT) and the improved rifamycin B producer (HP-130) were profiled at 5 time points. For each time point two biological replicates are provided.
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| Contributor(s) |
Peano C, Corti G, Talà A, Palumbo C, Siculella L, Damiano F, Forcato M, Malagoli Tagliazucchi G, Pasanisi D, Fuligni F, Pietrelli A, Bicciato S, De Bellis G, Alifano P |
| Citation(s) |
25149266 |
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| Submission date |
Sep 19, 2013 |
| Last update date |
Aug 27, 2014 |
| Contact name |
Silvio Bicciato |
| E-mail(s) |
[email protected]
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| Phone |
+39-049-827-6108
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| Organization name |
University of Padova
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| Department |
Molecular Medicine
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| Street address |
via U. Bassi 59/b
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| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
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| Platforms (1) |
| GPL17744 |
[AMEDS_expr] Custom Affymetrix Amycolatopsis mediterranei S699 10k chip |
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| Samples (20)
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| Relations |
| BioProject |
PRJNA219644 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE51015_RAW.tar |
23.0 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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