Transcriptome Analysis of an Antibiotic Downregulator Mutant and Synergistic Actinorhodin Stimulation via Disruption of a Precursor Flux Regulator in Streptomyces coelicolor

Abstract

Through microarray analysis of an antibiotic-downregulator-deleted Streptomyces coelicolor ⌬wblA ⌬SCO1712 mutant, 28 wblA-and SCO1712-dependent genes were identified and characterized. Among 14 wblAand SCO1712-independent genes, a carbon flux regulating 6-phosphofructokinase SCO5426 was additionally disrupted in the ⌬wblA ⌬SCO1712 mutant and further stimulated actinorhodin production in S. coelicolor, implying that both regulatory and precursor flux pathways could be synergistically optimized for antibiotic production.

Repression of Antibiotic Downregulator WblA by AdpA in Streptomyces coelicolor

“…3, real-time PCR analyses demonstrated that the levels of actII-orf4 transcripts were higher in the ⌬wblA mutant during the time course than in S. coelicolor M145 and the ⌬adpA mutant. It is consistent that the ⌬wblA mutant produced the highest level of actinorhodin and the ⌬adpA mutant did not produce actinorhodin at all (15,29,30). The levels of adpA transcripts from S. coelicolor M145 were similar to those in the ⌬wblA mutant, suggesting that the expression of adpA was not influenced by the wblA gene, and suggesting that the transcription of adpA occurs upstream of wblA transcription.…”

“…Based on these results, we wondered whether AdpA SC regulates wblA gene expression as an activator or repressor. To test this, transcription analysis was conducted with strains of S. coelicolor M145, the S. coelicolor ⌬wblA mutant (26,29), and the S. coelicolor ⌬adpA mutant (19). It was previously reported that the S. coelicolor ⌬wblA mutant produces a much higher level of actinorhodin than S. coelicolor M145 (29,30), whereas the adpA-deleted S. coelicolor strain does not produce actinorhodin at all in either liquid or plate cultures (15,16).…”

“…To test this, transcription analysis was conducted with strains of S. coelicolor M145, the S. coelicolor ⌬wblA mutant (26,29), and the S. coelicolor ⌬adpA mutant (19). It was previously reported that the S. coelicolor ⌬wblA mutant produces a much higher level of actinorhodin than S. coelicolor M145 (29,30), whereas the adpA-deleted S. coelicolor strain does not produce actinorhodin at all in either liquid or plate cultures (15,16). Each strain was grown in 100 ml of modified R5 (R5 without KH 2 PO 4 , CaCl 2 · 2H 2 O, L-proline, and growth factor) liquid medium, and total RNAs were isolated (RNeasy miniprep kit; Qiagen) at 8 h (before any antibiotic production from all strains), 25 h (when there is a slight production of actinorhodin from just the ⌬wblA mutant), and 35 h (when there is the production of actinorhodin from S. coelicolor M145 or the ⌬wblA mutant and no production of actinorhodin from the ⌬adpA mutant).…”

In silico aided metabolic engineering of Streptomyces roseosporus for daptomycin yield improvement

“…These data indicated that the effect of overexpressing zwf2 was slightly small when the three genes were overexpressed (Table S1). This might be due to the imperfect nature of the in silico metabolic model that could not account for complex regulatory mechanisms (Kim et al 2011). Besides, another reason might be the synergistic effect caused by the adjacent dptI and dptJ genes in the genome of S. roseosporus.…”

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana

“…Sequential targeted gene disruption of these two regulatory genes as well as SCO5426, a flux-controlling gene, resulted in a tripleknockout S. coelicolor mutant displaying significantly increased production of the polyketide antibiotic actinorhodin (15,16,46). To determine whether or not deletion of these regulatory genes could affect CsA hydroxylation activity in S. coelicolor, we introduced pMMBL312 into S. coelicolor mutants, generating S. coelicolor M145 ⌬SCO5426, S. coelicolor M145 ⌬wblA, and S. coelicolor M145 ⌬wblA ⌬SCO1712 ⌬SCO5426 (designated ESK616, ESK617, and ESK618, respectively).…”