TetR Family Transcriptional Regulator PccD Negatively Controls Propionyl Coenzyme A Assimilation in Saccharopolyspora erythraea

Xu, Zhen; Wang, Miaomiao; Ye, Bang‐Ce

doi:10.1128/jb.00281-17

Cited by 15 publications

(15 citation statements)

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“…It is worthwhile to note that genetic engineering of TFR elements has been shown to increase the yields of valuable antibiotics in industrial settings (13)(14)(15). Nonetheless, distinct from research related to TFRs for the biosynthesis of avermectin and erythromycin (13,(15)(16)(17)(18)(19), insights into the regulatory role of TFRs in lincomycin biosynthesis have not been reported. In the genome of S. lincolnensis LCGL, a derivative of S. lincolnensis LC-G with artificial synthetic 4ϫattB ⌽C31 (20), we identified 123 putative TFRs based on BLAST analysis with Pfam PF00440 (TetR_N) and the genome annotation of S. lincolnensis LC-G.…”

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confidence: 99%

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

et al. 2019

Appl Environ Microbiol

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Lincomycin A (Lin-A) is a widely used antibacterial antibiotic fermented by Streptomyces lincolnensis. However, the transcriptional regulatory mechanisms underlying lincomycin biosynthesis have seldom been investigated. Here, we first identified a TetR family transcriptional regulator (TFR), SLCG_2919, which negatively modulates lincomycin biosynthesis in S. lincolnensis LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lin cluster), including 25 structural genes, three resistance genes, and one regulatory gene, and to inhibit the transcription of these genes, demonstrating a directly regulatory role in lincomycin biosynthesis. Furthermore, we found that SLCG_2919 was not autoregulated, but directly repressed its adjacent gene, SLCG_2920, which encodes an ATP/GTP binding protein whose overexpression increased resistance against lincomycin and Lin-A yields in S. lincolnensis. The precise SLCG_2919 binding site within the promoter region of SLCG_2920 was determined by a DNase I footprinting assay and by electrophoretic mobility shift assays (EMSAs) based on base substitution mutagenesis, with the internal 10-nucleotide (nt) AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate that SLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in S. lincolnensis. The present study improves our understanding of molecular regulation for lincomycin biosynthesis. IMPORTANCE TetR family transcriptional regulators (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in Streptomyces species. However, knowledge of their function in lincomycin biosynthesis in S. lincolnensis remains unknown. The present study provides a new insight into the regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directly modulates lincomycin production and resistance. Intriguingly, SLCG_2919 and its adjoining gene, SLCG_2920, which encodes an ATP/GTP binding protein, were extensively distributed in diverse Streptomyces species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, dependent on the intervening AT-rich sequence rather than on the flanking inverted repeats found in the binding sites of other TFRs. These insights into transcriptional regulation of lincomycin biosynthesis by SLCG_2919 will be valuable in paving the way for genetic engineering of regulatory elements in Streptomyces species to improve antibiotic production.

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confidence: 99%

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

et al. 2019

Appl Environ Microbiol

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“…For an insertional deletion of the pccD gene in S. erythraea E3, a previously described homologous recombination strategy was used (29). Briefly, 1.5-kb DNA fragments upstream and downstream of the pccD gene locus were amplified from S. erythraea E3 genomic DNA by PCR using the primer pairs pUC3396upF/R and pUC3396dwF/R ( Table 2).…”

Section: Methodsmentioning

confidence: 99%

“…In this study, ΔgdhA1 and ΔBcdhE1 mutants demonstrated that the bkd operon plays an important role in BCAA degradation in S. erythraea. PccD was also demonstrated to negatively control the propionyl-CoA carboxylase genes (pccBCA) in S. erythraea (29). The pccBC and pccA operons were shown to have a major function in propionyl-CoA assimilation.…”

Section: Figmentioning

confidence: 99%

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PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production in Saccharopolyspora erythraea

Liu

2018

Appl Environ Microbiol

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Branched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis in In this study, we found that the operon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of the operon was upregulated 5-fold in a gene deletion strain (Δ strain) and decreased 3-fold in a overexpression strain (WT/pIB-), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion of significantly improved the Δ strain's growth rate, whereas overexpression repressed WT/pIB- growth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion of and the BcdhE1 gene (genes in the operon) resulted in lower growth rates of Δ and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that the operon is involved in BCAA degradation. Both overexpression (WT/pIB-) and inactivation (Δ strain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB- was decreased by 48%. Lastly, we explored the applications of engineering and in an industrial high-erythromycin-producing strain. deletion in industrial strain E3 (E3) improved erythromycin production by 20%, and the overexpression of in E3Δ (E3Δ/pIB-) increased erythromycin production by 39% compared with E3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strain E3. We describe a operon involved in BCAA degradation in The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield.

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“…One part was used to detect the dry cell weight (DCW) according to the previous method [40], and the other was used to analyze intracellular propionyl-CoA. Intracellular propionyl-CoA levels were detected using a modified method previously described by Xu et al [41]. Cells cultures were harvested and quenched in liquid nitrogen and washed twice with precooled phosphate-buffered saline (PBS, pH 8.0), then added with lysis buffer (10% trichloroacetic acid and 2 mM dithiothreitol), and repeatedly frozen and thawed four times using liquid nitrogen ice water, then sonicated on ice for 5 min (work 5 s, stop 5 s) using an ultrasonic disintegrator (JY96-IIN; Ningbo Xinzhi Instruments Inc., Ningbo, China).…”

Section: Determination Of Intracellular Propionyl-coa Concentrationsmentioning

confidence: 99%

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum

et al. 2020

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Background: Androstenedione (AD) is an important steroid medicine intermediate that is obtained via the degradation of phytosterols by mycobacteria. The production process of AD is mainly the degradation of the phytosterol aliphatic side chain, which is accompanied by the production of propionyl CoA. Excessive accumulation of intracellular propionyl-CoA produces a toxic effect in mycobacteria, which restricts the improvement of production efficiency. The 2-methylcitrate cycle pathway (MCC) plays a significant role in the detoxification of propionyl-CoA in bacterial. The effect of the MCC on phytosterol biotransformation in mycobacteria has not been elucidated in detail. Meanwhile, reducing fermentation cost has always been an important issue to be solved in the optimizing of the bioprocess. Results: There is a complete MCC in Mycobacterium neoaurum (MNR), prpC, prpD and prpB in the prp operon encode methylcitrate synthase, methylcitrate dehydratase and methylisocitrate lyase involved in MCC, and PrpR is a specific transcriptional activator of prp operon. After the overexpression of prpDCB and prpR in MNR, the significantly improved transcription levels of prpC, prpD and prpB were observed. The highest conversion ratios of AD obtained by MNR-prpDBC and MNR-prpR increased from 72.3 ± 2.5% to 82.2 ± 2.2% and 90.6 ± 2.6%, respectively. Through enhanced the PrpR of MNR, the in intracellular propionyl-CoA levels decreased by 43 ± 3%, and the cell viability improved by 22 ± 1% compared to MNR at 96 h. The nitrogen transcription regulator GlnR repressed prp operon transcription in a nitrogen-limited medium. The glnR deletion enhanced the transcription level of prpDBC and the biotransformation ability of MNR. MNR-prpR/ΔglnR was constructed by the overexpression of prpR in the glnR-deleted strain showed adaptability to low nitrogen. The highest AD conversion ratio by MNR-prpR/ΔglnR was 92.8 ± 2.7% at low nitrogen level, which was 1.4 times higher than that of MNR.

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TetR Family Transcriptional Regulator PccD Negatively Controls Propionyl Coenzyme A Assimilation in Saccharopolyspora erythraea

Cited by 15 publications

References 39 publications

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production in Saccharopolyspora erythraea

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum

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