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Biosynthesis method for side chain introducing amino acid source to polyketone skeleton and related genes

A technology of biosynthesis and polyketide skeleton, applied in biochemical equipment and methods, plant genetic improvement, botany equipment and methods, etc., can solve the problems of high preparation cost, low incorporation efficiency, limitations, etc.

Inactive Publication Date: 2015-05-06
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although in vitro synthesis of Stretcher Units (J. Am. Chem. Soc. 2001, 123, 5822-5823) and the use of acyl-NAC thioesters as Stretcher Unit substitutes (Nat. Prod. Rep. 2008, 25, 25-34) This dilemma can be somewhat alleviated, but the high preparation cost and extremely low incorporation efficiency still limit their application

Method used

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  • Biosynthesis method for side chain introducing amino acid source to polyketone skeleton and related genes
  • Biosynthesis method for side chain introducing amino acid source to polyketone skeleton and related genes
  • Biosynthesis method for side chain introducing amino acid source to polyketone skeleton and related genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] [Example 1] splenocin producing bacteria S.sp .CNQ431 genetic operating system establishment

[0072] 1. Donor Bacteria Preparation

[0073] Transform the plasmid containing the target into E. coli ET12567, pick a single colony on the plate and inoculate it into 5mL LB (containing the corresponding antibiotic) for overnight culture, pipette 4 mL of the bacterial solution into 50 mL LB (containing the corresponding antibiotic), place it in a shaker at 37 ℃, 250 rpm Cultivate until OD600=0.4 - 0.6. After centrifugation at 6000 rpm for 4 min, the bacteria were washed twice with 20 mL LB to remove antibiotics, and then resuspended in 1 mL LB as DNA donor bacteria.

[0074] 2. Recipient Bacteria Preparation

[0075] Take out a tube of spore suspension (6.7x109 cells / mL) stored at -80°C in 20% glycerol, centrifuge at 8000 rpm for 3 min to remove glycerol, and wash the spores twice with 0.5 mL TES buffer (0.05 M, pH 8.0) to Glycerol was removed, then resuspended with 50...

Embodiment 2

[0078] [Example 2] S.sp . Construction and screening of CNQ431 gene library

[0079] 1. Mini-digestion experiment

[0080] Add 10 u / μL of Alu ⅠRestriction endonuclease (Takara) was diluted with 10 × L buffer and sterilized water to make the working concentration (1×) of the buffer, and 15 μL of the enzyme concentration of 0.33 U / μL, and then prepare 250 μl of the reaction system (containing CNQ 431 gDNA 40 μl, 10× L buffer 25 μl), then divide the 250 μl reaction system into 1 × 50 μl and 7 × 25 μl, then take 2 μl and dilute to 0.33 u / μl in advance Alu Ⅰ Add 50 μl of the reaction system in the first tube, mix well and transfer 25 μl to the second tube of 25 μl, repeat this transfer step 7, all the above steps must be performed on ice, and then all the systems 37 After digestion at ℃ for 15 min, inactivation at 70 °C for 10 min, electrophoresis at 40 V, 0.5% agarose gel for 12 h in a cold room at 4 °C, staining with ethidium bromide, and observing the quality of enzyme dig...

Embodiment 3

[0093] [Example 3] Construction of mutant or recombinant strains

[0094] 1. SpnC gene disruption

[0095] Primer 5'-CG GAATTC GAGCGGCTGACCTCGTTCTG-3' and 5'-C CAAGCTT G

[0096] ACGACCGTGCACAGTCCGT-3'( EcoR I and Hind III restriction site is underlined) using the genome of CNQ431 as a template, a 3.08 kb middle fragment of the spnC gene was amplified and connected to PMD19-T, which was confirmed to be correct by sequencing. Bam HI- Hind III digestion, because the middle fragment of spnC has a Bam HI restriction site, so the excised fragment is only 1.1 kb, and then ligated into the plasmid pYH7 Bgl II- Hind III site. The constructed plasmid was named pWHU2406, and then transferred to CNQ431 through the genetic operating system. The grown binders were streaked onto MS plates to induce gene recombination. Pick the colony from the MS plate and inoculate it on the MS plate containing 50 μg / mL apramycin, culture at 30°C, and screen out Δ spnC The mutant stra...

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Abstract

The invention relates to a biosynthesis method for a side chain introducing amino acid source to a polyketone skeleton. The method is based on the biosynthetic pathway of an aroma extending unit of benzyl malonyl mono-acyl coenzyme A; the related genes of the biosynthesis and recognition of the extending unit come from the biosynthetic pathway of splenocin (SPN) and enterocin (ENC) in marine streptomyces Streptomycessp.CNQ431, and comprise genes enccP, enccH, spnE and spnD. The pathway first conducts deamination reduction carboxylation on phenylalanine to transform the phenylalanine into the benzyl malonyl mono-acyl coenzyme A extending unit, and then conducts selective acyltransferase recognition on the aroma and lards type extending units and uploads the extending units on the polyketone skeleton. The strategy and biosynthetic pathway directly transform amino acid into CoA polyketone extending unit and insert the extending unit to the polyketone carbon skeleton, greatly enrich diversified means and structural types of polyketone structure, and generate a positive promotion function on research and development of polyketone medicine.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a biosynthesis method for introducing side chains derived from amino acids into a polyketide skeleton in a polyketide synthesis system and genes related to biosynthesis. Background technique [0002] Polyketides (PKs) are a large class of important natural products widely derived from bacteria, fungi, and plants. Including macrolides, anthracyclines, polyethers, tetracyclines. Polyketides not only have various structures, but also have good biological and pharmaceutical activities (Angew Chem Int Ed Engl, 2009, 48(26): 4688-4716.). Such as antibacterial drugs (such as tetracycline, rifamycin), antifungal drugs (such as griseofulvin, amphotericin), antiparasitic drugs (such as avermycin, nemadectin), anticancer drugs (such as Drugs derived from polyketides such as doxorubicin, enediyne), anti-cholesterol drugs (such as lovastatin) have been used in the tr...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N15/60C12N15/54C12N15/52
Inventor 常晨晨黄荣邓子新瞿旭东
Owner WUHAN UNIV
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