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Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis

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ABSTRACT

Background: Milbemycins, a group of 16-membered macrolides with potent anthelminthic and insecticidal activity, are produced by several Streptomyces and used widely in agricultural, medical and veterinary fields. Milbemycin A3 and A4, the main components produced by Streptomyces bingchenggensis, have been developed as an acaricide to control mites. The subsequent structural modification of milbemycin A3/A4 led to other commercial products, such as milbemycin oxime, lepimectin and latidectin. Despite its importance, little is known about the regulation of milbemycin biosynthesis, which has hampered efforts to enhance milbemycin production via engineering regulatory genes.

Results: milR, a regulatory gene in the milbemycin (mil) biosynthetic gene cluster of S. bingchenggensis, encodes a large ATP-binding regulator of the LuxR family (LAL family), which contains an ATPase domain at its N-terminus and a LuxR-like DNA-binding domain at the C-terminus. Gene disruption and genetic complementation revealed that milR plays an important role in the biosynthesis of milbemycin. β-glucuronidase assays and transcriptional analysis showed that MilR activates the expression of the milA4-E operon and milF directly, and activates the other mil genes indirectly. Site-directed mutagenesis confirmed that the ATPase domain is indispensable for MilR’s function, and particularly mutation of the conserved amino acids K37A, D122A and D123A, led to the loss of MilR function for milbemycin biosynthesis. Overexpression of an extra copy of milR under the control of its native promoter significantly increased production of milbemycin A3/A4 in a high-producing industrial strain S. bingchenggensis BC04.

Conclusions: A LAL regulator, MilR, was characterized in the mil gene cluster of S. bingchenggensis BC04. MilR could activate milbemycin biosynthesis through direct interaction with the promoter of the milA4-E operon and that of milF. Overexpression of milR increased milbemycin A3/A4 production by 38 % compared with the parental strain BC04, suggesting that genetic manipulation of this activator gene could enhance the yield of antibiotics.

Electronic supplementary material: The online version of this article (doi:10.1186/s12934-016-0552-1) contains supplementary material, which is available to authorized users.

No MeSH data available.


Effect of milR disruption on Milbemycin A3/A4 production. a Genetic organization of mil gene cluster in Streptomyces bingchenggensis BC04 and diagram of milR disruption and complementation constructions. Each arrow indicates a separate open reading frame (ORF) and orientation of transcription. b HPLC analysis of milbemycin A3/A4 production in S. bingchenggensis BC04, BC04/pSET152, ΔmilR, ΔmilR/pSET152::milR. Absorbance at 242 nm was monitored
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Fig2: Effect of milR disruption on Milbemycin A3/A4 production. a Genetic organization of mil gene cluster in Streptomyces bingchenggensis BC04 and diagram of milR disruption and complementation constructions. Each arrow indicates a separate open reading frame (ORF) and orientation of transcription. b HPLC analysis of milbemycin A3/A4 production in S. bingchenggensis BC04, BC04/pSET152, ΔmilR, ΔmilR/pSET152::milR. Absorbance at 242 nm was monitored

Mentions: To determine the role of milR in milbemycin biosynthesis, a milR disruption mutant (ΔmilR) was constructed via homologous recombination (Fig. 2a). In ΔmilR, a 1753-bp fragment internal to milR was replaced by the kanamycin resistance gene, neo. The resulting ΔmilR was further confirmed by PCR (Additional file 1: Figure S1). ΔmilR was cultured in fermentation medium for 9 days and the production of milbemycin was tested. The result showed that no milbemycin was produced by ΔmilR in comparison with BC04 and BC04/pSET152 controls (Fig. 2b). To verify that the phenotype was the result of milR disruption, a complementation experiment was carried out, in which an integrating plasmid, pSET152::milR, was used to complement ΔmilR. In pSET152::milR, milR was driven by its own promoter. Milbemycin production was restored in the complemented strain (ΔmilR/pSET152::milR) (Fig. 2b). These results demonstrated that MilR is indispensable for milbemycin production in S. bingchenggensis.Fig. 2


Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis
Effect of milR disruption on Milbemycin A3/A4 production. a Genetic organization of mil gene cluster in Streptomyces bingchenggensis BC04 and diagram of milR disruption and complementation constructions. Each arrow indicates a separate open reading frame (ORF) and orientation of transcription. b HPLC analysis of milbemycin A3/A4 production in S. bingchenggensis BC04, BC04/pSET152, ΔmilR, ΔmilR/pSET152::milR. Absorbance at 242 nm was monitored
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Effect of milR disruption on Milbemycin A3/A4 production. a Genetic organization of mil gene cluster in Streptomyces bingchenggensis BC04 and diagram of milR disruption and complementation constructions. Each arrow indicates a separate open reading frame (ORF) and orientation of transcription. b HPLC analysis of milbemycin A3/A4 production in S. bingchenggensis BC04, BC04/pSET152, ΔmilR, ΔmilR/pSET152::milR. Absorbance at 242 nm was monitored
Mentions: To determine the role of milR in milbemycin biosynthesis, a milR disruption mutant (ΔmilR) was constructed via homologous recombination (Fig. 2a). In ΔmilR, a 1753-bp fragment internal to milR was replaced by the kanamycin resistance gene, neo. The resulting ΔmilR was further confirmed by PCR (Additional file 1: Figure S1). ΔmilR was cultured in fermentation medium for 9 days and the production of milbemycin was tested. The result showed that no milbemycin was produced by ΔmilR in comparison with BC04 and BC04/pSET152 controls (Fig. 2b). To verify that the phenotype was the result of milR disruption, a complementation experiment was carried out, in which an integrating plasmid, pSET152::milR, was used to complement ΔmilR. In pSET152::milR, milR was driven by its own promoter. Milbemycin production was restored in the complemented strain (ΔmilR/pSET152::milR) (Fig. 2b). These results demonstrated that MilR is indispensable for milbemycin production in S. bingchenggensis.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Milbemycins, a group of 16-membered macrolides with potent anthelminthic and insecticidal activity, are produced by several Streptomyces and used widely in agricultural, medical and veterinary fields. Milbemycin A3 and A4, the main components produced by Streptomyces bingchenggensis, have been developed as an acaricide to control mites. The subsequent structural modification of milbemycin A3/A4 led to other commercial products, such as milbemycin oxime, lepimectin and latidectin. Despite its importance, little is known about the regulation of milbemycin biosynthesis, which has hampered efforts to enhance milbemycin production via engineering regulatory genes.

Results: milR, a regulatory gene in the milbemycin (mil) biosynthetic gene cluster of S. bingchenggensis, encodes a large ATP-binding regulator of the LuxR family (LAL family), which contains an ATPase domain at its N-terminus and a LuxR-like DNA-binding domain at the C-terminus. Gene disruption and genetic complementation revealed that milR plays an important role in the biosynthesis of milbemycin. β-glucuronidase assays and transcriptional analysis showed that MilR activates the expression of the milA4-E operon and milF directly, and activates the other mil genes indirectly. Site-directed mutagenesis confirmed that the ATPase domain is indispensable for MilR’s function, and particularly mutation of the conserved amino acids K37A, D122A and D123A, led to the loss of MilR function for milbemycin biosynthesis. Overexpression of an extra copy of milR under the control of its native promoter significantly increased production of milbemycin A3/A4 in a high-producing industrial strain S. bingchenggensis BC04.

Conclusions: A LAL regulator, MilR, was characterized in the mil gene cluster of S. bingchenggensis BC04. MilR could activate milbemycin biosynthesis through direct interaction with the promoter of the milA4-E operon and that of milF. Overexpression of milR increased milbemycin A3/A4 production by 38 % compared with the parental strain BC04, suggesting that genetic manipulation of this activator gene could enhance the yield of antibiotics.

Electronic supplementary material: The online version of this article (doi:10.1186/s12934-016-0552-1) contains supplementary material, which is available to authorized users.

No MeSH data available.