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Biosynthetic gene cluster of maduramicin compound and application of biosynthetic gene cluster

A technology of biosynthesis and madomecin, which is applied in the field of bioengineering and can solve the problems of biosynthetic gene clusters without madomecin compounds

Active Publication Date: 2021-03-05
WUHAN HESHENG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, so far, there is no report on the study of gene clusters related to the biosynthesis of maduramicin compounds

Method used

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  • Biosynthetic gene cluster of maduramicin compound and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of maduramicin compound and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of maduramicin compound and application of biosynthetic gene cluster

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preparation example Construction

[0186] According to the method for preparing the maduramicin compound in the embodiment of the present invention, the maduramicin compound can be efficiently synthesized by using the aforementioned microorganisms for fermentation treatment. Then, through microbial wall breaking treatment and separation and purification treatment, the pure product of madomecin compound can be obtained efficiently, simply and at low cost.

[0187] According to some embodiments of the present invention, through gene prediction, the biosynthetic pathway of the madomecin compound is deduced as follows: image 3 . On the one hand, the extended synthesis of propionyl-CoA and methylmalonyl-CoA is catalyzed by the polyketide synthesis gene cluster to finally form a long-chain fatty acid chain. The intermediate product of madomecin is obtained, on the other hand, the glycosyl is catalyzed by genes such as madG1, madG2, madG3, madG4, madG5, madG6, madG7 and madG8, and then the glycosyl is transferred to...

Embodiment 1

[0191] Inoculate Actinomadura sp.J1-007 actinomycetes on the SFM plate and culture for about three to four days. When colonies are formed in the SFM medium, pick a single colony and culture it in the seed medium for about three to four days, and inoculate it into the fermentation medium according to the amount of 1% (the composition of the fermentation medium is 0.4% [w / v] Corn Syrup, 6% [w / v] Dextrose, 2.4% [w / v] Feather Meal, 0.005% [w / v] Fe 2 (SO 4 ) 3 , 0.3% [w / v] NaCl, 0.015% [w / v] K 2 HPO 4 ,0.1%[w / v]CaCO 3 , pH7.2. )middle. Culture conditions: 30 degrees, 220 rotation speed, culture for 7 to 8 days.

[0192] At day 7 or 8, the cultures were extracted with methanol, and the extracts were detected by LC-MS, using a commercial maduramycin standard as a positive control. Such as Figure 4 As shown, Actinomadura sp. J1-007 has the same retention time as the madurmicin standard (ammonium salt compound: m / z=934.16). The compound was further confirmed to be a madurami...

Embodiment 2

[0194] Taking Actinomadura sp.J1-007 as the research object, the sequence information of the biosynthetic gene cluster of maduramicin compounds was studied, and the specific methods were as follows:

[0195] 1. DNA extraction

[0196] (1) Streptomyces subtype Actinomadura sp. J1-007 was inoculated in TSBY medium and cultured for 2 days, and the cells were collected at 7000 rpm×5 min.

[0197] (2) Discard the supernatant of the collected bacteria, add 30mL SET buffer, break up, and centrifuge at 7000rpm for 5 minutes. Discard the supernatant again, add 10mL SET buffer, break up the cells, add 300μL lysozyme (100mg / ml), bathe in water at 37℃ for 30-60 minutes, add 100μL proteinase K (100mg / ml), 10μL RNase (10mg / ml ), in a 37°C water bath for 30 minutes.

[0198] (3) Add 1.2 mL of 10% SDS to the mixture obtained in step (2), bathe in water at 55°C for 2 hours, shake gently every 15 minutes until the liquid gradually becomes clear.

[0199] (4) Add 4 mL of 5M Nacl to the liquid o...

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Abstract

The invention discloses a biosynthetic gene cluster of a maduramicin compound and application of the biosynthetic gene cluster. The biosynthetic gene cluster of the maduramicin compound comprises an I-type linear polyketone synthetase gene module, a glycosyl synthesis related gene module, a modified gene module and a regulatory gene module, and the I-type linear polyketone synthetase gene module comprises a madA1 gene, a madA2 gene, a madA3 gene, a madA4 gene, a madA5 gene, a madA6 gene, a madA7 gene, a madA8 gene, a madA9 gene, a madA10 gene and a madA11 gene; the glycosyl synthesis related gene module comprises a madG1 gene, a madG2 gene, a madG3 gene, a madG4 gene, a madG5 gene, a madG6 gene, a madG7 gene and a madG8 gene; the modified gene module comprises a madO1 gene, a madO2 gene, amadI gene, a madE gene, a madP1 gene, a madP2 gene, a madM1 gene, a madM2 gene, a madG9 gene, a madG10 gene and a madTE gene; and the regulatory gene module comprises a madR1 gene, a madR2 gene, a madR3 gene and a madR4 gene.

Description

technical field [0001] The present invention relates to the field of bioengineering, in particular, to a biosynthetic gene cluster of a madomecin compound and applications thereof, more specifically, to a biosynthetic gene cluster of a madomecin compound, microorganisms and methods for preparing a madomecin compound method. Background technique [0002] Coccidiosis is an intestinal infectious disease of wild and domestic animals caused by coccidia, characterized by poor growth, impaired performance and even death in infected animals. The global poultry industry is the most affected, with losses estimated at more than $3 billion per year. Polyether ionophores are lipophilic chelating agents that have the effect of transporting cations across cell membranes to damage cells. Maduramicin, which shows potent anticoccidial activity, is produced by the bacterium Actinomadura yumaensis through aerobic fermentation and was first identified in 1983 with an effective dose of 5 mg / kg,...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/53C12N15/60C12N15/55C12N15/61C12N15/31C12N15/74C12N1/21C12P17/18C12R1/01
CPCC12N15/52C12N9/1029C12N9/0004C12N9/88C12N9/90C12N9/1007C12N9/16C12N9/1048C12N9/0071C12N9/14C12N9/1241C07K14/195C12N15/74C12P17/181C12Y203/01187Y02A50/30
Inventor 刘然
Owner WUHAN HESHENG TECH CO LTD
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