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Journal of Bacteriology, August 2008, p. 5587-5596, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00592-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification and Utility of FdmR1 as a Streptomyces Antibiotic Regulatory Protein Activator for Fredericamycin Production in Streptomyces griseus ATCC 49344 and Heterologous Hosts{triangledown} ,{dagger}

Yihua Chen,1 Evelyn Wendt-Pienkowski,1 and Ben Shen1,2,3*

Division of Pharmaceutical Sciences,1 University of Wisconsin National Cooperative Drug Discovery Group,2 Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53705-22223

Received 29 April 2008/ Accepted 6 June 2008

The fredericamycin (FDM) A biosynthetic gene cluster, cloned previously from Streptomyces griseus ATCC 49344, contains three putative regulatory genes, fdmR, fdmR1, and fdmR2. Their deduced gene products show high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family (FdmR1) or to MarR-like regulators (FdmR and FdmR2). Here we provide experimental data supporting FdmR1 as a SARP-type activator. Inactivation of fdmR1 abolished FDM biosynthesis, and FDM production could be restored to the fdmR1::aac(3)IV mutant by expressing fdmR1 in trans. Reverse transcription-PCR transcriptional analyses revealed that up to 26 of the 28 genes within the fdm gene cluster, with the exception of fdmR and fdmT2, were under the positive control of FdmR1, directly or indirectly. Overexpression of fdmR1 in S. griseus improved the FDM titer 5.6-fold (to about 1.36 g/liter) relative to that of wild-type S. griseus. Cloning of the complete fdm cluster into an integrative plasmid and subsequent expression in heterologous hosts revealed that considerable amounts of FDMs could be produced in Streptomyces albus but not in Streptomyces lividans. However, the S. lividans host could be engineered to produce FDMs via constitutive expression of fdmR1; FDM production in S. lividans could be enhanced further by overexpressing fdmC, encoding a putative ketoreductase, concomitantly with fdmR1. Taken together, these studies demonstrate the viability of engineering FDM biosynthesis and improving FDM titers in both the native producer S. griseus and heterologous hosts, such as S. albus and S. lividans. The approach taken capitalizes on FdmR1, a key activator of the FDM biosynthetic machinery.

* Corresponding author. Mailing address: Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, 777 Highland Ave., Madison, WI 53705-2222. Phone: (608) 263-2673. Fax: (608) 262-5345. E-mail: bshen{at}pharmacy.wisc.edu

{triangledown} Published ahead of print on 13 June 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, August 2008, p. 5587-5596, Vol. 190, No. 16
0021-9193/08/$08.00+0     doi:10.1128/JB.00592-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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