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Recombinant streptomyces lydicus for producing cellulase and natamycin and application

A technology of Streptomyces lidi and cellulase, applied in the field of bioengineering, can solve the problems of narrow antibacterial spectrum, single antibacterial mechanism, limited types of metabolites, etc., and achieve the effect of improving the ability to degrade cellulose

Inactive Publication Date: 2015-04-01
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a natural strain, the metabolites of a single strain are limited, and there will be a single antibacterial mechanism, poor antibacterial ability, and narrow antibacterial spectrum.

Method used

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  • Recombinant streptomyces lydicus for producing cellulase and natamycin and application
  • Recombinant streptomyces lydicus for producing cellulase and natamycin and application
  • Recombinant streptomyces lydicus for producing cellulase and natamycin and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, construct the recombinant Streptomyces lydides producing cellulase and natamycin

[0083] 1. Preparation of DNA fragments

[0084] The synthetic nucleotide sequence is the DNA fragment of SEQ ID No.2 in the sequence listing. Among them, the DNA fragment shown in SEQ ID No.2, its name is P-glu-vhb, the DNA fragment from the 5' end to the 3' end is: the recognition site of Nde I, the coding sequence of cellulase, The recognition site of Xba I, the promoter of erythromycin resistance gene, the coding sequence of hemoglobin and the recognition site of EcoR I. SEQ ID No.2 is made up of 2140 nucleotides, the 1st-6th nucleotide of SEQ ID No.2 is the recognition site of Nde I, and the 7th-1488th nucleotide of SEQ ID No.2 is The coding sequence of cellulase, the 1489-1494th nucleotide of SEQ ID No.2 is the recognition site of Xba I, and the 1501-1691th nucleotide of SEQ ID No.2 is the erythromycin resistance gene In the promoter, nucleotides 1692-2132 of SEQ ID ...

Embodiment 2

[0100] Embodiment 2, the cellulase activity of the recombinant Streptomyces lydidii producing cellulase and natamycin

[0101] The experiment was repeated three times, and the results of each repeated experiment were as follows:

[0102] Cellulase-producing and natamycin-producing recombinant Streptomyces lydidis spores (5×10 7 1) Add 50mL cellulase production medium (cellulase production medium consists of peptone, CMC-Na (sodium carboxymethylcellulose), NH 4 NO 3 、K 2 HPO 4 , NaCl and water, among which peptone, CMC-Na, NH 4 NO 3 、K 2 HPO 4 , NaCl mass percentages are respectively 2%, 1%, 0.4%, 0.01%, 0.5%), at 29 ℃, 220rpm, cultivated for 20 hours to obtain the recombinant Streptomyces lydidi culture fluid, the recombinant Streptomyces lydidi culture solution was added to 100mL cellulase production medium and diluted to OD 600nm was 0.1, and continued to cultivate at 29° C. and 220 rpm for 12 hours to obtain a 12-hour recombinant Streptomyces lydidis fermentation b...

Embodiment 3

[0107] Embodiment 3, the ability of recombinant Streptomyces lydidae to produce natamycin

[0108] One, the preparation of natamycin fermented liquid

[0109] The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:

[0110] Inoculate the recombinant Streptomyces lydida producing cellulase and natamycin in Example 1 on the Gaoshi No. 1 slant medium, cultivate it at 28°C for 7-10 days, and treat it to produce a sufficient amount of spores. The spores were scraped and inoculated into 50ml of seed medium in a 250ml Erlenmeyer flask in 2-3 rings, placed on a temperature-controllable shaker, and incubated at a constant temperature for 24h-30h under the conditions of 28°C and 200rpm (rotation radius: 13mm); Then it was inoculated under aseptic conditions in the fermentation medium first (YSG substratum) in 60 500ml Erlenmeyer flasks (every bottle of liquid volume is 100ml), and the OD of each bottle was inoculated. 600 The values...

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Abstract

The invention discloses recombinant streptomyces lydicus for producing cellulase and natamycin and an application. A construction method of recombinant streptomyces lydicus comprises steps as follows: expression vectors of an encoding gene of hemoglobin and an encoding gene of cellulase are guided into streptomyces lydicus to obtain the recombinant streptomyces lydicus for producing cellulase and natamycin; hemoglobin is protein of A 1) or A 2), in A 1), the amino acid sequence of hemoglobin is represented as SEQ ID No.3 in a sequence table, and in A 2), hemoglobin is protein which has the hemoglobin function and is derived from A 1); and cellulase is protein of B 1) or B 2), in B 1), the amino acid sequence of cellulase is represented as SEQ ID No.4 in the sequence table, and in B 2), cellulase is protein which has the cellulase function and is derived from B 1). The expression vectors comprises an erythromycin resistance gene promoter, the encoding gene of cellulase, an erythromycin resistance gene promoter and the encoding gene of hemoglobin in the direction from upstream to downstream.

Description

technical field [0001] The invention relates to a cellulase- and natamycin-producing recombinant Streptomyces lydidis and its application in the field of bioengineering. Background technique [0002] With the increase of world population and the decrease of arable land, food supply is becoming more and more severe. However, the perennial occurrence of crop diseases and insect pests has brought huge losses to the world economy (Chen Jun et al., 2010). my country is a large agricultural country based on planting. The use of pesticides can reduce economic losses by about 30 billion yuan per year. Among them, the amount of chemical pesticide application accounts for more than 80% of the amount of pesticide use, which causes serious environmental pollution, pesticide residues and other problems, and seriously threatens human health and the ecological environment. Therefore, biological control is an inevitable trend in the development of pesticides. Among them, Streptomyces is w...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N1/21C12N9/42C12P19/62A01N63/00A01N63/02A01P3/00C12R1/465
Inventor 吴慧玲刘伟成李锦锦刘霆董丹张涛涛卢彩鸽张殿朋刘德文田兆丰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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