A GATA-type transcription factor AcAREB for nitrogen metabolism is involved in regulation of cephalosporin biosynthesis in Acremonium chrysogenum

Guan, Feifei; Pan, Yuanyuan; Li, Jinyang; Liu, Gang

doi:10.1007/s11427-017-9118-9

Cited by 14 publications

(9 citation statements)

References 19 publications

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“…For routine subcloning, Escherichia coli DH5α and JM109 were grown at 37 °C in Luria–Bertani (LB) medium supplemented with ampicillin or apramycin when necessary. E. coli BL21(DE3) was used for protein expression [ 10 , 11 ]. The methylation-deficient E. coli ET12567 ( dam - dcm - hsds - ) was used to propagate non-methylated DNA.…”

Section: Methodsmentioning

confidence: 99%

“…The EMSAs were performed as previously described with some modifications [ 11 ]. The probe P E27 containing a 159 bp upstream region of SCO3127 was amplified with the primers 3127EF/3127ER.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNAs were isolated from strains grown in YEME medium as described previously [ 11 ]. Quality and quantity of RNAs were examined by UV spectroscopy and checked by agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

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SCO3129, a TetR family regulator, is responsible for osmotic stress in Streptomyces coelicolor

Pan

et al. 2018

Synthetic and Systems Biotechnology

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Streptomyces are the soil-dwelling bacteria with a complex lifecycle and a considerable ability to produce a variety of secondary metabolites. Osmoregulation is important for their lifecycle in nature. In the genome of Streptomyces coelicolor M145, SCO3128 (encodes a putative fatty acid desaturase), SCO3129 (encodes a putative TetR family regulator) and SCO3130 (encodes a putative l-carnitine dehydratase) constitute a transcriptional unit, and its transcript was found to be in response to osmotic stress. Disruption of SCO3130 led to a bald phenotype on MMG medium and the mycelia lysis on the edge of the colony when KCl/NaCl was added to the medium. These results indicated that SCO3130 is important for the osmotic stress resistance in S. coelicolor. Transcriptional analysis and electrophoretic mobility shift assays (EMSA) demonstrated that SCO3129 repressed the transcription of SCO3128-3130 operon through directly binding to the promoter region of SCO3128, indicating that SCO3129 regulates the transcription of SCO3128-3130 in response to osmotic stress.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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SCO3129, a TetR family regulator, is responsible for osmotic stress in Streptomyces coelicolor

Pan

et al. 2018

Synthetic and Systems Biotechnology

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“…Finally, they were verified by sequencing (GENEWIZ, China). Reverse transcription PCR (RT-PCR) and real-time RT-PCR were performed as described previously [ 21 , 22 ].…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Acremonium chrysogenum for improving cephalosporin C production independent of methionine stimulation

Gao

Pan

et al. 2018

Microb Cell Fact

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BackgroundCephalosporin C (CPC) produced by Acremonium chrysogenum is one of the most important drugs for treatment of bacterial infectious diseases. As the major stimulant, methionine is widely used in the industrial production of CPC. In this study, we found methionine stimulated CPC production through enhancing the accumulation of endogenous S-adenosylmethionine (SAM). To overcome the methionine dependent stimulation of CPC production, the methionine cycle of A. chrysogenum was reconstructed by metabolic engineering.ResultsThree engineered strains were obtained by overexpressing the SAM synthetase gene AcsamS and the cystathionine-γ-lyase gene mecB, and disrupting a SAM dependent methyltransferase gene Acppm1, respectively. Overexpression of AcsamS resulted in fourfold increase of CPC production which reached to 129.7 µg/mL. Disruption of Acppm1 also increased CPC production (up to 135.5 µg/mL) through enhancing the accumulation of intracellular SAM. Finally, an optimum recombinant strain (Acppm1DM-mecBOE) was constructed through overexpressing mecB in the Acppm1 disruption mutant. In this strain, CPC production reached to the maximum value (142.7 µg/mL) which was 5.5-fold of the wild-type level and its improvement was totally independent of methionine stimulation.ConclusionsIn this study, we constructed a recombinant strain in which the improvement of CPC production was totally independent of methionine stimulation. This work provides an economic route for improving CPC production in A. chrysogenum through metabolic engineering.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0936-5) contains supplementary material, which is available to authorized users.

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“…Total RNA was isolated using Trizol Reagent (Invitrogen, USA) according to the commercial protocol and digested by DNase I to remove the genomic DNA as described previously [ 24 , 25 ]. cDNA was obtained using the PrimeScript™ RT Reagent Kit (TaKaRa).…”

Section: Methodsmentioning

confidence: 99%

Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum

Wang

et al. 2018

Microb Cell Fact

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BackgroundAutophagy is used for degradation of cellular components and nutrient recycling. Atg8 is one of the core proteins in autophagy and used as a marker for autophagic detection. However, the autophagy of filamentous fungi is poorly understood compared with that of Saccharomyces cerevisiae. Our previous study revealed that disruption of the autophagy related gene Acatg1 significantly enhanced cephalosporin C yield through reducing degradation of cephalosporin biosynthetic proteins in Acremonium chrysogenum, suggesting that modulation of autophagic process is one promising way to increase antibiotic production in A. chrysogenum.ResultsIn this study, a S. cerevisiae ATG8 homologue gene Acatg8 was identified from A. chrysogenum. Acatg8 could complement the ATG8 mutation in S. cerevisiae, indicating that Acatg8 is a functional homologue of ATG8. Microscope observation demonstrated the fluorescently labeled AcAtg8 was localized in the cytoplasm and autophagosome of A. chrysogenum, and the expression of Acatg8 was induced by nutrient starvation. Gene disruption and genetic complementation revealed that Acatg8 is essential for autophagosome formation. Disruption of Acatg8 significantly reduced fungal conidiation and delayed conidial germination. Localization of GFP-AcAtg8 implied that autophagy is involved in the early phase of conidial germination. Similar to Acatg1, disruption of Acatg8 remarkably enhanced cephalosporin C yield. The cephalosporin C biosynthetic enzymes (isopenicillin N synthase PcbC and isopenicillin N epimerase CefD2) and peroxisomes were accumulated in the Acatg8 disruption mutant (∆Acatg8), which might be the main reasons for the enhancement of cephalosporin C production. However, the biomass of ΔAcatg8 decreased drastically at the late stage of fermentation, suggesting that autophagy is critical for A. chrysogenum cell survival under nutrition deprived condition. Disruption of Acatg8 also resulted in accumulation of mitochondria, which might produce more reactive oxygen species (ROS) which promotes fungal death. However, the premature death is unfavorable for cephalosporin C production. To solve this problem, a plasmid containing Acatg8 under control of the xylose/xylan-inducible promoter was introduced into ∆Acatg8. Conidiation and growth of the recombinant strain restored to the wild-type level in the medium supplemented with xylose, while the cephalosporin C production maintained at a high level even prolonged fermentation.ConclusionsOur results demonstrated inducible expression of Acatg8 and disruption of Acatg8 remarkably increased cephalosporin C production. This study provides a promising approach for yield improvement of cephalosporin C in A. chrysogenum.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1021-9) contains supplementary material, which is available to authorized users.

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A GATA-type transcription factor AcAREB for nitrogen metabolism is involved in regulation of cephalosporin biosynthesis in Acremonium chrysogenum

Cited by 14 publications

References 19 publications

SCO3129, a TetR family regulator, is responsible for osmotic stress in Streptomyces coelicolor

SCO3129, a TetR family regulator, is responsible for osmotic stress in Streptomyces coelicolor

Metabolic engineering of Acremonium chrysogenum for improving cephalosporin C production independent of methionine stimulation

Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum

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