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Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production.

Carata E, Peano C, Tredici SM, Ferrari F, Talà A, Corti G, Bicciato S, De Bellis G, Alifano P - Microb. Cell Fact. (2009)

Bottom Line: In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected.In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants.At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy. alifano@ilenic.unile.it.

ABSTRACT

Background: There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.

Results: Spontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.

Conclusion: Rifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

No MeSH data available.


Related in: MedlinePlus

Semi-quantitative analysis of the SACE 1456-SACE 1459-specific transcripts by RT real time PCR. (A) Genetic map of the SACE 1456-SACE 1459 genetic cluster coding for enzymes involved in the valine catabolic pathway (B). (B) Possible metabolic pathways leading to propionyl-CoA and 2-methylmalonil-CoA, the two building blocks for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B. (C) Semi-quantitative analysis of SACE 1456-SACE 1459-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.
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Figure 6: Semi-quantitative analysis of the SACE 1456-SACE 1459-specific transcripts by RT real time PCR. (A) Genetic map of the SACE 1456-SACE 1459 genetic cluster coding for enzymes involved in the valine catabolic pathway (B). (B) Possible metabolic pathways leading to propionyl-CoA and 2-methylmalonil-CoA, the two building blocks for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B. (C) Semi-quantitative analysis of SACE 1456-SACE 1459-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.

Mentions: This cluster comprises 13 genes that were up-regulated in rif1, and down-regulated in the rif6 (Figure 5, right down-panel). The most important members of this cluster are genes encoding NADH dehydrogenase I (nuoC [SACE 6900], nuoD [SACE 6899], nuoE [SACE 6898], nuoF [SACE 6897]), and genes coding for key enzymes of a major feeder pathway of erythromycin biosynthesis (mmsA2 [SACE 1456] coding for methylmalonate semialdehyde dehydrogenase, SACE 1457 coding for acyl-CoA dehydrogenase-like activity). In particular SACE 1456 and SACE 1457 are the first two genes of a putative operon also including SACE 1458 (echA9) encoding putative enoyl-CoA hydratase/isomerase and SACE 1459 (mmsB) coding for 3-hydroxyisobutyrate dehydrogenase (Figure 6A).


Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production.

Carata E, Peano C, Tredici SM, Ferrari F, Talà A, Corti G, Bicciato S, De Bellis G, Alifano P - Microb. Cell Fact. (2009)

Semi-quantitative analysis of the SACE 1456-SACE 1459-specific transcripts by RT real time PCR. (A) Genetic map of the SACE 1456-SACE 1459 genetic cluster coding for enzymes involved in the valine catabolic pathway (B). (B) Possible metabolic pathways leading to propionyl-CoA and 2-methylmalonil-CoA, the two building blocks for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B. (C) Semi-quantitative analysis of SACE 1456-SACE 1459-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667423&req=5

Figure 6: Semi-quantitative analysis of the SACE 1456-SACE 1459-specific transcripts by RT real time PCR. (A) Genetic map of the SACE 1456-SACE 1459 genetic cluster coding for enzymes involved in the valine catabolic pathway (B). (B) Possible metabolic pathways leading to propionyl-CoA and 2-methylmalonil-CoA, the two building blocks for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B. (C) Semi-quantitative analysis of SACE 1456-SACE 1459-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.
Mentions: This cluster comprises 13 genes that were up-regulated in rif1, and down-regulated in the rif6 (Figure 5, right down-panel). The most important members of this cluster are genes encoding NADH dehydrogenase I (nuoC [SACE 6900], nuoD [SACE 6899], nuoE [SACE 6898], nuoF [SACE 6897]), and genes coding for key enzymes of a major feeder pathway of erythromycin biosynthesis (mmsA2 [SACE 1456] coding for methylmalonate semialdehyde dehydrogenase, SACE 1457 coding for acyl-CoA dehydrogenase-like activity). In particular SACE 1456 and SACE 1457 are the first two genes of a putative operon also including SACE 1458 (echA9) encoding putative enoyl-CoA hydratase/isomerase and SACE 1459 (mmsB) coding for 3-hydroxyisobutyrate dehydrogenase (Figure 6A).

Bottom Line: In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected.In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants.At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy. alifano@ilenic.unile.it.

ABSTRACT

Background: There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.

Results: Spontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.

Conclusion: Rifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

No MeSH data available.


Related in: MedlinePlus