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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 overexpression of milR on antibiotics production and cell dry weight. a Milbemycin production in S. bingchenggensis BC04 and BC04::milR. b Cell dry weight of S. bingchenggensis BC04 and BC04::milR. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations, *P < 0.05
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Fig6: Effect of overexpression of milR on antibiotics production and cell dry weight. a Milbemycin production in S. bingchenggensis BC04 and BC04::milR. b Cell dry weight of S. bingchenggensis BC04 and BC04::milR. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations, *P < 0.05

Mentions: Overexpression of transcriptional activators is an efficient approach to increase production of antibiotics, especially engineered activators under the control of strong constitutive promoters. Using this strategy, nikkomycin production in Streptomyces ansochromogenes and oxytetracycline biosynthesis in Streptomyces rimosus were enhanced significantly by introducing an extra copy of activator genes sanG (driven by constitutive hrdB promoter) and otcR (controlled by the constitutive SF14 promoter), respectively [30, 31]. In this work, milR was confirmed to be a direct activator of milbemycin biosynthesis (Fig. 2); thus, engineering the expression levels of milR might be a rational strategy to improve milbemycin production in S. bingchenggensis BC04, a high-producing industrial strain for milbemycin A3/A4 production. First, milR was placed under the control of a strong constitutive hrdB promoter in an integrative plasmid (pSET152) and in a multicopy plasmid (pKC1139). These two recombinant plasmids were transformed into BC04 to create BC04::hrdBmilR and BC04::hrdBmilRs, respectively. Unexpectedly, milbemycin A3/A4 production in BC04::hrdBmilR or BC04::hrdBmilRs was lower compared with that of BC04 (Additional file 5: Figure S5). Meanwhile, cell growth rate of these two strains was reduced in seed medium. BC04::hrdBmilRs exhibited a slower growth rate than that of BC04::hrdBmilR. These results indicated that overexpression of milR under a strong constitutive promoter did not increase milbemycin production. A single copy of milR with its own promoter was then cloned into pSET152 to generate pSET152::milR. This plasmid was transformed into BC04, resulting in BC04::milR. Notably, in BC04::milR, the final production of milbemycin A3/A4 increased by 38 %, reaching 4069 mg/l, compared with 2947 mg/l in BC04 (Fig. 6a). We also determined the biomass of BC04 and BC04::milR. The results showed that they had comparable growth rates and final biomass, and that the increase in milbemycin A3/A4 production was attributed to the overexpression of milR (Fig. 6b).Fig. 6


Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis
Effect of overexpression of milR on antibiotics production and cell dry weight. a Milbemycin production in S. bingchenggensis BC04 and BC04::milR. b Cell dry weight of S. bingchenggensis BC04 and BC04::milR. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations, *P < 0.05
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Related In: Results  -  Collection

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Fig6: Effect of overexpression of milR on antibiotics production and cell dry weight. a Milbemycin production in S. bingchenggensis BC04 and BC04::milR. b Cell dry weight of S. bingchenggensis BC04 and BC04::milR. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations, *P < 0.05
Mentions: Overexpression of transcriptional activators is an efficient approach to increase production of antibiotics, especially engineered activators under the control of strong constitutive promoters. Using this strategy, nikkomycin production in Streptomyces ansochromogenes and oxytetracycline biosynthesis in Streptomyces rimosus were enhanced significantly by introducing an extra copy of activator genes sanG (driven by constitutive hrdB promoter) and otcR (controlled by the constitutive SF14 promoter), respectively [30, 31]. In this work, milR was confirmed to be a direct activator of milbemycin biosynthesis (Fig. 2); thus, engineering the expression levels of milR might be a rational strategy to improve milbemycin production in S. bingchenggensis BC04, a high-producing industrial strain for milbemycin A3/A4 production. First, milR was placed under the control of a strong constitutive hrdB promoter in an integrative plasmid (pSET152) and in a multicopy plasmid (pKC1139). These two recombinant plasmids were transformed into BC04 to create BC04::hrdBmilR and BC04::hrdBmilRs, respectively. Unexpectedly, milbemycin A3/A4 production in BC04::hrdBmilR or BC04::hrdBmilRs was lower compared with that of BC04 (Additional file 5: Figure S5). Meanwhile, cell growth rate of these two strains was reduced in seed medium. BC04::hrdBmilRs exhibited a slower growth rate than that of BC04::hrdBmilR. These results indicated that overexpression of milR under a strong constitutive promoter did not increase milbemycin production. A single copy of milR with its own promoter was then cloned into pSET152 to generate pSET152::milR. This plasmid was transformed into BC04, resulting in BC04::milR. Notably, in BC04::milR, the final production of milbemycin A3/A4 increased by 38 %, reaching 4069 mg/l, compared with 2947 mg/l in BC04 (Fig. 6a). We also determined the biomass of BC04 and BC04::milR. The results showed that they had comparable growth rates and final biomass, and that the increase in milbemycin A3/A4 production was attributed to the overexpression of milR (Fig. 6b).Fig. 6

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. &beta;-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&rsquo;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&nbsp;% 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.