• KSBB Journal
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• v.24 no.3
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• pp.259-266
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• 2009

AfsR2 in Streptomyces lividans, a 63-amino acid protein with limited sequence homology to Streptomyces sigma factors, has been known for a global regulatory protein stimulating multiple antibiotic biosynthetic pathways. Although the detailed regulatory mechanism of AfsK-AfsR-AfsR2 system has been well characterized, very little information about the AfsR2-dependent down-stream regulatory genes were characterized. Recently, the null mutant of afsS in S. coelicolor (the identical ortholog of afsR2) has been characterized through DNA microarray system, revealing that afsS deletion regulated several genes involved in antibiotic biosynthesis as well as phosphate-starvation. Through comparative DNA microarray analysis of afsR2-overexpressed S. lividans, here we also identify several afsR2-dependent genes involved in phosphate starvation, morphological differentiation, and antibiotic regulation in S. lividans, confirming that the AfsR2 plays an important pleiotrophic regulatory role in Streptomyces species.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.248-253
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• 2005

AfsR2, originally identified from Streptomyces lividans, is a global regulatory protein which stimulates antibiotic biosynthesis. Through its stable chromosomal integration, the high level of gene expression of afsR2 significantly induced antibiotic production as well as the sporulation of S. lividans, implying the presence of yet-uncharacterized AfsR2-target proteins. To identify and evaluate the putative AfsR2-target proteins involved in antibiotic regulation, the proteomics-driven approach was applied to the wild-type S. lividans and the afsR2-integrated actinorhodin overproducing strain. The 20 gel-electrophoresis gave approximately 340 protein spots showing different protein expression patterns between these two S. lividans strains. Further MALDI-TOF analysis revealed several AfsR2-target proteins, including glyceraldehyde-3-phosphate dehydrogenase, putative phosphate transport system regulator, guanosine penta phosphate synthetase/polyribonucleotide nucleotidyltransferase, and superoxide dismutase, which suggests that the AfsR2 should be a pleiotropic regulatory protein which controls differential expressions of various kinds of genes in Streptomyces species.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1477-1480
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• 2006

The 63-amino-acid-encoding afsR2 is a global antibiotics-stimulating regulatory gene identified from the chromosome of Streptomyces lividans. To dissect a putative functional domain in afsR2, several afsR2-derivative deletion constructs were generated and screened for the loss of actinorhodin-stimulating capability. The afsR2-derivative construct missing a 50-bp C-terminal region significantly lost its actinorhodin-stimulating capability in S. lividans. In addition, site-directed mutagenesis on amino acid positions of #57-#61 in a 50-bp C-terminal region, some of which are conserved among known Sigma 70 family proteins, significantly changed the AfsR2's activity. These results imply that the C-terminal region of AfsR2 is functionally important for antibiotics-stimulating capability and the regulatory mechanism might be somehow related to the sigma-like domain present in the C-terminal of AfsR2.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.672-680
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• 2015

As a global regulatory gene in Streptomyces, afsR can activate the biosynthesis of many secondary metabolites. The effect of afsR on the biosynthesis of a phenazine metabolite, lomofungin, was studied in Streptomyces lomondensis S015. There was a 2.5-fold increase of lomofungin production in the afsR-overexpressing strain of S. lomondensis S015 N1 compared with the wild-type strain. Meanwhile, the transcription levels of afsR and two important genes involved in the biosynthesis of lomofungin (i.e., phzC and phzE) were significantly upregulated in S. lomondensis S015 N1. The optimization of metal chlorides was investigated to further increase the production of lomofungin in the afsR-overexpressing strain. The addition of different metal chlorides to S. lomondensis S015 N1 cultivations showed that CaCl₂, FeCl₂, and MnCl₂ led to an increase in lomofungin biosynthesis. The optimum concentrations of these metal chlorides were obtained using response surface methodology. CaCl₂ (0.04 mM), FeCl₂ (0.33 mM), and MnCl₂ (0.38 mM) gave a maximum lomofungin production titer of 318.0 ± 10.7 mg/l, which was a 4.1-fold increase compared with that of S. lomondensis S015 N1 without the addition of a metal chloride. This work demonstrates that the biosynthesis of phenazine metabolites can be induced by afsR. The results also indicate that metal chlorides addition might be a simple and useful strategy for improving the production of other phenazine metabolites in Streptomyces.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.68-72
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• 2002

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin is present in the two closely related bacterial species, Streptomyces lividans and Streptomyces coelicolor, it normally is expressed only in S. coelicolor---generating the deep blue colonies responsible for the S. coelicolor name. However, multiple copies of the afsR2 gene, which activates actinorhodin synthesis, result in the ability of S. lividansto also synthesize large amounts of actinorhodin. Here we report that the phenotypic property that historicially distinguishes these two Streptomycesspecies is determined conditionally by the carbon source used for culture. Whereas growth on glucose repressed actinorhodin production in S. lividans, culture on solid media containing glycerol as the sole carbon source dramatically increased the expression of afsR2 mRNA---leading to extensive actinorhodin synthesis by S. lividansand obliterating its phenotypic distinction from S. coelicolor. afsR2 transcription under these conditions was developmentally regulated, rising sharply at the time of aerial mycelium formation and coinciding temporally with the onset of actinorhodin production. Our results, which identify media-dependent parallel pathways that regulate actinorhodin synthesis in S. lividans, demonstrate carbon source control of actinorhodin production through the regulation of afsR2 mRNA synthesis. The nucleotide sequences of afsR2 revealed two putative important domains; the domain containing direct repeats in the middle and the domain homologous to sigma factor sequence in the C-terminal end. In this work, we constructed various sized afsR2-derivatives and compared the actinorhodin stimulating effects in S. lividans TK21. The experimental data indicate that the domain homologous to sigma factor sequence in the C-terminal end of afsR2 plays a critical role as an antibiotic stimulating function. In addition, we also observed that the single copy integration of afsR2 regulatory gene into S. lividans TK21 chromosome significantly activates antibiotic overproduction.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.577-581
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• 2003

Streptomyces lividans is one of the most commonly-used streptomyetes strain as a molecular cloning and expression host. Unlike its close relative S. coelicolor, however, S. lividans rarely produces secondary metabolite such as actinorhodin in a typical glucose-containing culture condition due to insufficient expression of some antibiotic regulatory genes including afsR2. Although multiple copies of afsR2 or a glycerol-specific culture condition stimulated actinorhodin production in S. lividans, both failed to stimulate actinorhodin production in S. lividans cultured in a typical glucose-containing medium. To generate a culture-condition-independent actinorhodin-overproducing S. lividans strain the afsR2 gene was integrated into the S. lividans TK21 chromosome via homologous recombination, followed by the genetic confirmation. This S. lividans strain produced a significant amount of actinorhodin in both glucose-containing liquid and plate cultures, with higher actinorhodin productivity compared to the S. lividans containing multiple copies of afsR2. These results suggest that a chromosomal integration of a single copy of an antibiotic regulatory gene is a promising method for the development of a stable antibiotic-overproducing streptomycetes strain.

• Journal of Microbiology and Biotechnology
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• v.19 no.2
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• pp.121-127
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• 2009

Sequencing analysis of a 5-kb DNA fragment from Streptomyces venezuelae ATCC 15439 revealed the presence of one 3.1-kb open reading frame(ORF), designated as afsR-sv. The deduced product of afsR-sv(1,056 aa) was found to have high homology with the global regulatory protein AfsR. Homology-based analysis showed that aftR-sv represents a transcriptional activator belonging to the Streptomyces antibiotic regulatory protein(SARP) family that includes an N-terminal SARP domain containing a bacterial transcriptional activation domain(BTAD), an NB-ARC domain, and a C-terminal tetratricopeptide repeat domain. Gene expression analysis by reverse transcriptase PCR(RT-PCR) demonstrated the activation of transcription of genes belonging to pikromycin production, when aftR-sv was overexpressed in S. venezuelae. Heterologous expression of the aftR-sv in different Streptomyces strains resulted in increased production of the respective antibiotics, suggesting that afsR-sv is a positive regulator of antibiotics biosynthesis.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1089-1092
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• 2004

An actinorhodin nonproducing Streptomyces lividans was converted to an actinorhodin overproducer through a single chromosomal integration of an antibiotic regulatory gene, afsR2. This strain exhibited early actinorhodin production and an average of 37.5% higher productivity than the S. lividans containing multiple copies of afsR2 plasmid in a glucose-containing liquid culture.

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.211-215
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• 2006

AfsR2 is a global regulatory protein involved in the stimulation of secondary metabolite biosynthesis in various Streptomyces species including avermectin-producing S. avermitilis. Among several AfsR2-dependent genes identified from the comparative proteomics, the polyribonucleotide nucleotidyltransferase (PNP) and the glyceraldehyde-3-phosphate dehydrogenase (GPD) genes were previously proposed to regulate the actinorhodin production in S. lividans upon afsR2 over-expression positively and negatively, respectively. To show the biological significance of the PNP and GPD genes in the S. avermitilis strains, these two genes were functionally expressed in both the wild-type and the avermectin-overproducing mutant strains. The PNP gene expression stimulated secondary metabolite production in the wild-type S. avermitilis ATCC31267, but not in the avermectin-overproducing S. avermitilis ATCC31780. Interestingly, the GDP gene expression stimulated secondary metabolite production by 4-fold in the wild-type S. avermitilis ATCC31267 and by 2.5-fold in the avermectin-overproducing S. avermitilis ATCC31780, respectively. These results suggest that the biological significance of the afsR2-dependent PNP and GPD gene expressions on antibiotic biosynthetic regulation could be significantly different depending on Streptomyces species.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.39 no.5
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• pp.527-534
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• 2015

This paper presents an integrated chassis control with electronic stability control (ESC) and active front steering (AFS) under lateral force constraint on AFS. The control yaw moment is calculated using a sliding mode control. The tire forces generated by ESC and AFS are determined using weighted pseudo-inverse based control allocation (WPCA) in order to generate the control yaw moment. On a low friction road, AFS is not effective when the lateral tire forces of front wheels are easily saturated. To solve problem, the lateral force of AFS is limited to its maximum and the braking of ESC is applied with WPCA. To evaluate the effectiveness of the proposed method, a simulation was performed on the vehicle simulation package, $CarSim^{(R)}$. From the simulation, it was verified that the proposed method could enhance the maneuverability and lateral stability if the lateral force of AFS exceeds its maximum.