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Genetic engineering bacterium for producing sugar-free ramoplanin, its construction method and application thereof

A technology of genetically engineered bacteria and genes, applied in the field of recombinant shuttle vectors and transformants, to achieve good industrialization prospects and increase stability

Inactive Publication Date: 2014-07-09
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology improves upon previous methods used to make artificial yeast cell walls without containing harmful substances like antibiotics. It also produces less sugars than traditional ones while maintaining their original properties. By creating these modified cells through specific techniques, they have improved characteristics such as increased production levels over time when compared to regular yeasts due to better control over metabolism pathways. Overall, this new approach allows us to improve the efficiency and quality of producing natural products from biomass resources more efficiently.

Problems solved by technology

The technical problem addressed in this patents relates to finding ways to prevent resistence caused by certain types of microbicides called remopolonins from causing harmful effects when administered externally into humans due to their ability to bind with other molecules like sugars found naturally occurring within the body's digestion system. This binding leads to reduced effectiveness over time given these substances have been developed specifically towards treatments aim at killing germinary cells involved in various medical conditions including inflammatory bowel disease.

Method used

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  • Genetic engineering bacterium for producing sugar-free ramoplanin, its construction method and application thereof
  • Genetic engineering bacterium for producing sugar-free ramoplanin, its construction method and application thereof
  • Genetic engineering bacterium for producing sugar-free ramoplanin, its construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning of double exchanged left and right arms

[0040] In Actinoplanes sp.ATCC 33076, ramo-orf26, ramo-orf27 and ramo-orf28 are sequentially upstream of ramo-orf29, ramo-orf30 and ramo-orf31 are sequentially downstream of ramo-orf29, we take ramo The fragment from -orf9 upstream to ramo-orf26, the 5' end fragment of ramo-orf29 are used as the left arm of the double crossover, and the 3' end fragment of the ramo-orf29 gene to ramo-orf30 is used as the right arm of the double crossover to construct a double crossover plasmid . Firstly, primers were designed according to the published corresponding sequence of Actinoplanes sp.ATCC33076 (US 7635765):

[0041] Upstream of the left arm is 5'-AAA AAGCTT CAGGGCGATGAGGATGC-3';

[0042] Downstream of the left arm is 5'-AAA TCTAGA GAAGCCGAAGACGCG-3';

[0043] Upstream of the right arm is 5'-AAA TCTAGA GCGGTCTCGCTGCTCT-3';

[0044] Downstream of the right arm is 5'-AAA GATATC GCTGCTCGACGTAGGC-3'.

[0045] ...

Embodiment 2

[0046] Example 2: Construction of double-exchange site-specific in-frame knockout of ramo-orf29 plasmid pCJS1004

[0047] The right arm in Example 1 was connected to the plasmid pKC1139 (purchased from Shanghai Bioengineering Technology Service Co., Ltd.), and the access sites XbaI and EcoRV were inserted to obtain the plasmid, which was named pCJS1003. Connect the left arm of Example 1 into the plasmid pCJS1003 in the forward direction, and access the sites HindIII and XbaI to obtain the plasmid. Transform the ligated product into Escherichia coli DH5α. Use the ampicillin-resistant LB plate to pick positive clones, and in LB medium After culturing overnight at 37°C, a small amount of plasmid was extracted for enzyme digestion and PCR verification, and a double-crossover in-frame knockout plasmid was finally constructed, named pCJS1004. Build a strategy map see figure 2 .

Embodiment 3

[0048] Example 3: Construction of Actinoplanes sp.ATCC 33076.D29, a sugar-free remoplanin-producing genetically engineered bacterium with knockout of glycosyltransferase

[0049] Pick an appropriate amount of bacteria from the slant of Actinomycetes mobilis ATCC 33076 and culture them in 50ml TSB medium for about 56 hours to reach the logarithmic growth phase. Transfer 1% (v / v) inoculum to 50ml TSB medium and culture them for 48 hours Let the bacteria reach the late logarithmic growth period, centrifuge and pour off the supernatant to obtain mycelia, wash the mycelium twice with 20ml LB liquid medium (4000rpm, 10min, 4°C), and finally resuspend in 20ml LB medium In, ready to use. Transform competent Escherichia coli ET12567 with pCJS1004, pick the transformants to 4ml LB medium [containing abramycin (Am) 100 μg / ml, kanamycin (Kn) 25 μg / ml, chloramphenicol (Cm) 100 μg / ml] in a small test tube, shake culture at 37°C for 12 hours. Escherichia coli ET12567 was inoculated in a 25...

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Abstract

The invention discloses a genetic engineering bacterium for producing sugar-free ramoplanin, which is the genetic engineering bacterium for inhibiting or knocking the glycosyltransferase gene for producing ramoplanin out in actinoplanes wild strain. The invention also discloses a preparation method of the genetic engineering bacterium and as well as a used recombinant shuttle vector and a transformant, and an application of the genetic engineering bacterium for producing sugar-free ramoplanin on production of sugar-free ramoplanin or a f sugar-free ramoplanin derivative.

Description

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Claims

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Application Information

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Owner SHANGHAI INST OF PHARMA IND
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