Genetically engineered bacterium capable of increasing yield of spinosads as well as construction method and application

A technology of genetically engineered bacteria and spinosyn, which is applied in the field of genetically engineered bacteria with high spinosyn production and its construction, and can solve problems such as unbalanced spinosyn synthesis pathways and the like

Active Publication Date: 2014-04-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above studies ignored the imbalance of the spinosyn synthesis pathway

Method used

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  • Genetically engineered bacterium capable of increasing yield of spinosads as well as construction method and application
  • Genetically engineered bacterium capable of increasing yield of spinosads as well as construction method and application
  • Genetically engineered bacterium capable of increasing yield of spinosads as well as construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, gene element cloning and plasmid construction containing corresponding gene element

[0085] Digest the pIB139 plasmid containing the ermE* promoter (SEQ ID NO.23) with HindIII and XbaI. The enzyme used is Fermentas Fast Taq restriction endonuclease. The enzyme digestion system is as follows: 30ng of the target DNA fragment, add 2μL of 10×buffer , 1 μL each of restriction enzymes HindIII and XbaI (endonucleases are added at the end), and supplemented with distilled water to 20 μL. The enzyme digestion system was placed at 37°C for 30 min to obtain a 0.3 kb HindIII / XbaI fragment containing the ermE* promoter. This HindIII / XbaI fragment was inserted into plasmid pOJ260, resulting in pOJ261 plasmid.

[0086] Using the primer pair spnK-F-Ndel (SEQ ID NO.1) and spnK-R-Xbal (SEQ ID NO.2) from the chromosome of Saccharopolyspora spinosa (Saccharopolyspora spinosa) ATCC49460 (hereinafter referred to as Saccharopolyspora spinosa ATCC49460) The spnK gene sequence ...

Embodiment 2

[0099] Embodiment 2, the construction of genetically engineered bacteria LU101, LU102 and LU104 that can improve spinosad output:

[0100]All constructed plasmids were introduced into S. spinosa from Escherichia coli S17-1 by combined transfer, and integrated into the chromosome by homologous recombination. The steps are as follows:

[0101] The plasmid pLU101 was used to transform competent Escherichia coli S17-1, and the obtained Escherichia coli was screened on an LB plate for apramycin resistance (ampramycin 100 μg / mL). The transformants were picked and cultured in 4 mL LB (ampramycin 100 μg / ml) with shaking at 37°C for 12 hours.

[0102] Pick an appropriate amount of Saccharopolyspora spinosa ATCC49460 from the slope and culture it in TSB liquid medium for about 72 hours at 30°C, transfer 1% of the inoculum to 50mL TSB and culture it for about 42 hours to make the bacterial liquid reach the late logarithmic growth period, centrifuge and pour off the supernatant , the cel...

Embodiment 3

[0129] Example 3, Fermentation Verification of Genetically Engineered Bacteria That Can Improve Spinosad Production

[0130] Strain medium:

[0131] Slant culture medium (g / L): casein peptone 2.5, glucose 5, beef extract 3, agar 20, magnesium sulfate 2, the balance is water, pH=7.5 before sterilization.

[0132] Seed medium (g / L): Glucose 10, N-Z-Amine A (acid hydrolyzed casein) 30, yeast extract 1.0, beef extract 1.0, MgSO42.0, the balance is water, pH=7.3 before sterilization.

[0133] Fermentation medium (g / L): glucose 68, milk peptone 25, cottonseed protein 22, corn steep liquor 14.5, methyl oleate 40, calcium carbonate 5, the balance is water, pH=7.5 before sterilization.

[0134] Shake flask fermentation culture: wash the mature spores with appropriate amount of sterile normal saline on the fresh plates of the genetically engineered bacteria LU101, LU102, LU104 that can increase the production of spinosad and the wild bacteria Saccharopolymona spinosa ATCC49460, and was...

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Abstract

The invention discloses a genetically engineered bacterium capable of increasing the yield of spinosads as well as a construction method and an application. The construction method of the genetically engineered bacterium capable of increasing the yield of spinosads comprises the following steps: (1) inserting an spnk expression cassette into an escherichia coli-saccharopolyspora spinosa shuttle plasmid pOJ260 to construct a recombinant plasmid pLU101; (2) guiding the recombinant plasmid pLU101 into saccharopolyspora spinosa ATCC49460, and integrating the recombinant plasmid with a genome through homologous recombination, thus obtaining the genetically engineered bacterium capable of increasing the yield of spinosads and naming the genetically engineered bacterium LU101. The genetically engineered bacterium constructed by the method can increase the yield of spinosads.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a genetically engineered bacterium with high spinosad production and a construction method thereof. Background technique [0002] Spinosyn is a 21-carbon macrolide compound produced by the fermentation of the actinomycete Saccharopolyspora spinosa, which contains two deoxysugars: 3-O-methylated rhamnose and fultosamine . Spinosyns A and D are the two major components in S. spinosa fermentation broth. Spinosyns have shown broad-spectrum insecticidal activity and are bio-efficient insecticides that are environmentally friendly and have no harm to mammals. Spinosad green pesticides were awarded the 1999 Presidential Green Chemicals Challenge Award for their low risk, no specific target species, low environmental impact, and low toxicity to humans and animals. [0003] The spinosyn synthesis pathway is encoded by 27 genes. Five genes (SpnA, spnB, spnC, spnD, and spnE) encode type I p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12P19/62C12R1/01
Inventor 卢文玉薛超友张香梅
Owner TIANJIN UNIV
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