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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.


MilR directly activates promoters of milA4-E, orf1 and milF. a MilR-regulating regions are indicated by two vertical green arrows in the mil gene cluster. b Chromogenic assays on AS-1 agar plates containing the substrate 5-bromo-4-chloro-3-indolyl-β-d-glucoronide. Data are representative of three independent experiments
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Fig3: MilR directly activates promoters of milA4-E, orf1 and milF. a MilR-regulating regions are indicated by two vertical green arrows in the mil gene cluster. b Chromogenic assays on AS-1 agar plates containing the substrate 5-bromo-4-chloro-3-indolyl-β-d-glucoronide. Data are representative of three independent experiments

Mentions: milR is situated in the middle of the mil gene cluster; therefore, it is possible that the transcription of some mil genes might be regulated by MilR. To determine the potential targets of MilR, first, co-transcription analysis was performed to confirm the putative operons. Total RNAs were extracted from S. bingchenggensis BC04 after the onset of milbemycin production (3 days of cultivation in fermentation medium) and used as templates for cDNA synthesis and reverse transcription polymerase chain reaction (RT-PCR) analysis. Primers flanking four intergenic regions (regions 1–4) within the mil gene cluster were used: generation of PCR-amplified products by these primers would indicate transcription across the intergenic region (Additional file 2: Figure S2). The results showed that the mil gene cluster contains four transcriptional units (milA2-C, milA4-E, milR-A3 and milA1-D) and two individually transcribed genes (milF and orf1). Based on the above results, the promoters of the six transcriptional units were then cloned separately upstream of gusA [encoding β-glucuronidase (GUS)]. The resulting plasmids were integrated into the ФC31 attB site of Streptomyces coelicolor M1146. At the same time, the coding region of milR was cloned downstream of PhrdB in pIJ10500 to generate pIJ10500::PhrdBmilR. pIJ10500::PhrdBmilR was subsequently integrated into the ФBT1 attB site of the S. coelicolor M1146 derivatives containing the six different reporter constructs. Then gusA transcriptional fusions were assessed in agar-based chromogenic assays using 5-bromo-4-chloro-3-indolyl-β-d-glucuronide as the substrate (Fig. 3). None of the strains containing mil promoter::gusA plasmids that lacked constitutively expressed MilR gave GUS activity. When MilR was constitutively expressed, transcription of gusA from PmilA4, Porf1 and PmilF was readily detected; however, transcription of gusA from PmilA2, PmilR and PmilA1 was not detected. These results indicated that the promoters of milA4-E operon, orf1 and milF, are probably the direct targets of MilR.Fig. 3


Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis
MilR directly activates promoters of milA4-E, orf1 and milF. a MilR-regulating regions are indicated by two vertical green arrows in the mil gene cluster. b Chromogenic assays on AS-1 agar plates containing the substrate 5-bromo-4-chloro-3-indolyl-β-d-glucoronide. Data are representative of three independent experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015266&req=5

Fig3: MilR directly activates promoters of milA4-E, orf1 and milF. a MilR-regulating regions are indicated by two vertical green arrows in the mil gene cluster. b Chromogenic assays on AS-1 agar plates containing the substrate 5-bromo-4-chloro-3-indolyl-β-d-glucoronide. Data are representative of three independent experiments
Mentions: milR is situated in the middle of the mil gene cluster; therefore, it is possible that the transcription of some mil genes might be regulated by MilR. To determine the potential targets of MilR, first, co-transcription analysis was performed to confirm the putative operons. Total RNAs were extracted from S. bingchenggensis BC04 after the onset of milbemycin production (3 days of cultivation in fermentation medium) and used as templates for cDNA synthesis and reverse transcription polymerase chain reaction (RT-PCR) analysis. Primers flanking four intergenic regions (regions 1–4) within the mil gene cluster were used: generation of PCR-amplified products by these primers would indicate transcription across the intergenic region (Additional file 2: Figure S2). The results showed that the mil gene cluster contains four transcriptional units (milA2-C, milA4-E, milR-A3 and milA1-D) and two individually transcribed genes (milF and orf1). Based on the above results, the promoters of the six transcriptional units were then cloned separately upstream of gusA [encoding β-glucuronidase (GUS)]. The resulting plasmids were integrated into the ФC31 attB site of Streptomyces coelicolor M1146. At the same time, the coding region of milR was cloned downstream of PhrdB in pIJ10500 to generate pIJ10500::PhrdBmilR. pIJ10500::PhrdBmilR was subsequently integrated into the ФBT1 attB site of the S. coelicolor M1146 derivatives containing the six different reporter constructs. Then gusA transcriptional fusions were assessed in agar-based chromogenic assays using 5-bromo-4-chloro-3-indolyl-β-d-glucuronide as the substrate (Fig. 3). None of the strains containing mil promoter::gusA plasmids that lacked constitutively expressed MilR gave GUS activity. When MilR was constitutively expressed, transcription of gusA from PmilA4, Porf1 and PmilF was readily detected; however, transcription of gusA from PmilA2, PmilR and PmilA1 was not detected. These results indicated that the promoters of milA4-E operon, orf1 and milF, are probably the direct targets of MilR.Fig. 3

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.