Amycolatopsis mediterranei S699
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PROVIDER: PRJNA219644 | ENA |
REPOSITORIES: ENA
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Project description:Comparative Genomics Reveals New Molecular targets for Accelerated Improvement of Erythromycin-producing Strains
| PRJNA144545 | ENA
Project description:Information about molecular mechanisms underlying improvement of antibiotic-producing microorganisms with traditional mutation and screening approach is largely missing. This information is essential to develop rational approaches to strain improvement. In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of an erythromycin overproducer, which was obtained by the traditional mutate-and-screen method. Compared with parental Saccharopolyspora erythraea NRRL 2338, a total of 117 deletion, 78 insertion and 12 transposition sites were found across the genome of the overproducer. Among them, 71 insertion/deletion sites mapped within coding sequences (CDSs) generating frame-shift mutations. Moreover, single nucleotide variations affecting a total of 144 CDSs were identified between the two genomes. Overall, variations affect a total of 227 proteins in the genome of the overproducer. The analysis of metabolic pathways demonstrated that a considerable number of mutations affect genes coding for key enzymes involved in central carbon and nitrogen metabolism, and biosynthesis of secondary metabolites, redirecting common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, may affect global expression profile. Consistent transcriptomic data were obtained from a comparative analysis between NRRL 2338 and the erythromycin overproducer gene expression profiling performed with DNA microarray. Genomic data also indicated that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes. Our findings, largely unanticipated, reveal new targets suitable for rationale improvement of antibiotic-producing strains. According to a rough estimate, actinomycetes, which are among the most abundant bacteria in soil, produce over 70% of naturally occurring antibiotics and other biologically active substances including anti-tumour agents and immunosuppressants. However, these microorganisms must often be genetically improved for higher production before they can be used in the industry. Strain improvement has traditionally relied on multiple rounds of random mutagenesis and screening. This method is essentially empirical, and notably time-consuming and expansive. Currently, the availability of molecular genetics tools and useful information about the biosynthetic pathways and genetic control for most of biologically active substances has opened the way for improving strains by rational engineering. These rational strain improvement strategies benefit of the support of genomic and post-genomic technologies. In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of an erythromycin overproducer, which was obtained by the traditional mutate-and-screen method. Our findings, largely unanticipated, reveal new molecular targets suitable for rationale improvement of antibiotic-producing strains by recombinant technologies, and support the evidence that combining classical and recombinant strain improvement with a solid fermentation development program is the best way to improve production of biologically active substances. Erythromycin over-producing Strain at various time points
2012-05-23 | E-GEOD-30600 | biostudies-arrayexpress
Project description:Amycolatopsis mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.
| S-EPMC7332547 | biostudies-literature
Project description:Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1-11) and seven known analogues (12-18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4-11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 μg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 μM, respectively.
| S-EPMC7563148 | biostudies-literature
Project description:Rifamycin W, the most predominant intermediate in the biosynthesis of rifamycin, needs to undergo polyketide backbone rearrangement to produce rifamycin B via an oxidative cleavage of the C-12/C-29 double bond. However, the mechanism of this putative oxidative cleavage has not been characterized yet. Rif-Orf5 (a putative cytochrome P450 monooxygenase) was proposed to be involved in the cleavage of this olefinic moiety of rifamycin W. In this study, the mutant strain Amycolatopsis mediterranei S699 Δrif-orf5 was constructed by in-frame deleting the rif-orf5 gene to afford thirteen rifamycin W congeners (1-13) including seven new ones (1-7). Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Presumably, compounds 1-4 were derivatized from rifamycin W via C-5/C-11 retro-Claisen cleavage, and compounds 1-3, 9 and 10 featured a hemiacetal. Compounds 5-7 and 11 showed oxygenations at various sites of the ansa chain. In addition, compounds 1-3 exhibited antibacterial activity against Staphylococcus aureus with minimal inhibitory concentration (MIC) values of 5, 40 and 0.5 µg/mL, respectively. Compounds 1 and 3 showed modest antiproliferative activity against HeLa and Caco-2 cells with half maximal inhibitory concentration (IC50) values of about 50 µM.
| S-EPMC8301457 | biostudies-literature
Project description:Information about molecular mechanisms underlying improvement of antibiotic-producing microorganisms with traditional mutation and screening approach is largely missing. This information is essential to develop rational approaches to strain improvement. In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of an erythromycin overproducer, which was obtained by the traditional mutate-and-screen method. Compared with parental Saccharopolyspora erythraea NRRL 2338, a total of 117 deletion, 78 insertion and 12 transposition sites were found across the genome of the overproducer. Among them, 71 insertion/deletion sites mapped within coding sequences (CDSs) generating frame-shift mutations. Moreover, single nucleotide variations affecting a total of 144 CDSs were identified between the two genomes. Overall, variations affect a total of 227 proteins in the genome of the overproducer. The analysis of metabolic pathways demonstrated that a considerable number of mutations affect genes coding for key enzymes involved in central carbon and nitrogen metabolism, and biosynthesis of secondary metabolites, redirecting common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, may affect global expression profile. Consistent transcriptomic data were obtained from a comparative analysis between NRRL 2338 and the erythromycin overproducer gene expression profiling performed with DNA microarray. Genomic data also indicated that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes. Our findings, largely unanticipated, reveal new targets suitable for rationale improvement of antibiotic-producing strains. According to a rough estimate, actinomycetes, which are among the most abundant bacteria in soil, produce over 70% of naturally occurring antibiotics and other biologically active substances including anti-tumour agents and immunosuppressants. However, these microorganisms must often be genetically improved for higher production before they can be used in the industry. Strain improvement has traditionally relied on multiple rounds of random mutagenesis and screening. This method is essentially empirical, and notably time-consuming and expansive. Currently, the availability of molecular genetics tools and useful information about the biosynthetic pathways and genetic control for most of biologically active substances has opened the way for improving strains by rational engineering. These rational strain improvement strategies benefit of the support of genomic and post-genomic technologies. In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of an erythromycin overproducer, which was obtained by the traditional mutate-and-screen method. Our findings, largely unanticipated, reveal new molecular targets suitable for rationale improvement of antibiotic-producing strains by recombinant technologies, and support the evidence that combining classical and recombinant strain improvement with a solid fermentation development program is the best way to improve production of biologically active substances.
2012-05-24 | GSE30600 | GEO
Project description:Amycolatopsis mediterranei Raw sequence reads
| PRJNA1091067 | ENA
Project description:The genus Amycolatopsis is well known for its ability to produce antibiotics, and an increasing number of valuable biotechnological applications, such as bioremediation, biodegradation, bioconversion, and potentially biofuel, that use this genus have been developed. Amycolatopsis mediterranei is an industrial-scale producer of the important antibiotic rifamycin, which plays a vital role in antimycobacterial therapy. Genetic studies of Amycolatopsis species have progressed slowly due to the lack of efficient transformation methods and stable plasmid vectors. In A. mediterranei U32, electroporation and replicable plasmid vectors have been developed. Here, we establish a simple and efficient conjugal system by transferring integrative plasmid pDZL802 from ET12567 (pUZ8002) to A. mediterranei U32, with an efficiency of 4 × 10-5 CFU per recipient cell. This integrative vector, based on the ?BT1 int-attP locus, is a stable and versatile tool for A. mediterranei U32, and it may also be applicable to various other Amycolatopsis species for strain improvement, heterologous protein expression, and synthetic biology experiments.
| S-EPMC5639847 | biostudies-literature
Project description:Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.
| S-EPMC3187414 | biostudies-literature