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FK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned fromStreptomyces sp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes,tcs2 ,tcs7 , andfkbN , which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion oftcs2 did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting thattcs2 is not involved in their biosynthesis.fkbN overexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion offkbN caused the complete loss of FK506 and FK520 production. Although the overexpression oftcs7 decreased the levels of FK506 and FK520 production slightly, a deletion oftcs7 caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally,fkbN overexpression in thetcs7 deletion strain resulted in a 4.0-fold (21 mg liter−1 ) increase in FK506 production compared to that by the wild-type strain. This suggests thatfkbN encodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product oftcs7 negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated byfkbN in a positive manner and negatively bytcs7 .

Roles of fkbN in Positive Regulation and tcs7 in Negative Regulation of FK506 Biosynthesis in Streptomyces sp. Strain KCTC 11604BP