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Method for constructing engineering strain with high yield of FK520 and strain with high yield of Streptomyces hygroscopicus

A technology of FK520 and engineering strains, which is applied in the field of biotechnology engineering and can solve the problems of insignificant effect of component optimization of FK520, increase of cost of separation and purification steps, and reduction of fermentation titer of FK520.

Active Publication Date: 2018-01-26
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existence of FK523 reduces the fermentation titer of FK520 due to shunting effect on the one hand, on the other hand, its structural similarity also causes difficulty to the separation of FK520 pure product, has increased the cost of separation and purification steps, so it is urgent to improve the fermentation efficiency of FK520. The proportion of components
However, since FK520 and FK523 share the same biosynthetic pathway, the usual genetic manipulation methods have little effect on the component optimization of FK520

Method used

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  • Method for constructing engineering strain with high yield of FK520 and strain with high yield of Streptomyces hygroscopicus
  • Method for constructing engineering strain with high yield of FK520 and strain with high yield of Streptomyces hygroscopicus
  • Method for constructing engineering strain with high yield of FK520 and strain with high yield of Streptomyces hygroscopicus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Construction of Ethylmalonyl-CoA Biosynthesis Gene Expression Plasmid pSETerm-pnP5P6

[0102] After extensive screening, the nucleotide sequence (accession number) containing the biosynthetic gene of ethylmalonyl-CoA was obtained (accession number: JQ624413), and the plasmid pUC57- pn (synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.).

[0103] The pnP5 and pnP6 gene amplification primers were synthesized respectively, and the pnP5 gene and the pnP6 gene were respectively amplified by PCR.

[0104] The primers used to amplify pnP5 are:

[0105] PNP5-152-For: TATCATATGTTTGGCGTCAAGCATGGAAC (SEQ ID NO: 1)

[0106] PNP5-152-Rev: TATTCTAGAGTCCTTCGAGTCGTTTCATG (SEQ ID NO: 2)

[0107] The primers used to amplify pnP6 are:

[0108] PNP6-152-For: TATCATATGCGGTTCGCACTTCGCATC (SEQ ID NO: 3)

[0109] PNP6-152-Rev: TATTCTAGACCTCAACTGCTGTGGCTGTG (SEQ ID NO: 4)

[0110] The pnP5 gene and pnP6 gene were respectively placed under the control of the strong promoter ermEp* by...

Embodiment 2

[0112] Intrageneric conjugative transfer of plasmid pSETerm-pnP5P6 from Escherichia coli to the starting species Streptomyceshygroscopicus var.ascomyceticus ATCC 14891

[0113] Preparation of conjugative transfer donor bacteria: pick a single colony from the E. coli culture plate containing plasmid pSETerm-pnP5P6, transfer it to a test tube containing 2-3 mL LB, and culture it with shaking at 37°C overnight, draw 1 mL of the bacterial liquid and transfer it to 50 mL LB, Place in a shaker at 37°C at 250rpm and culture to OD 600 0.4-0.5. The bacterial solution was centrifuged, washed twice with an equal volume of LB, collected in a 1.5mL test tube, and then resuspended with 1mL of LB as the donor bacteria.

[0114] Preparation of conjugative transfer recipient bacteria: The spore suspension of ATCC 14891 bacteria stored at -80°C in 20% glycerol was washed twice with TES buffer, then resuspended with 500 μL TES buffer, heat-shocked at 50°C for 10 minutes, and then added Mix 500...

Embodiment 3

[0117] Genotype verification of genetically engineered strains

[0118] The zygotes with apramycin resistance were picked to culture in TSB medium containing apramycin, cultured at 28°C for 3-4 days, and the cells were collected to extract genomic DNA. Since pnP5 and pnP6 are exogenously introduced genes, the engineered strains can be verified by directly amplifying the target gene fragments by PCR. Two pairs of primers were designed for the pnP5 and pnP6 genes respectively, and the primer sequences were verified by PCR as follows:

[0119] The primers used to verify the pnP5 gene are:

[0120] PNP5-yz-For: TGGCGTCAAGCATGGAAC (SEQ ID NO:5)

[0121] PNP5-yz-Rev: TCCTTCGAGTCGTTTCATG (SEQ ID NO: 6)

[0122] The primers used to verify the pnP6 gene are:

[0123] PNP6-yz-For: CGGTTCGCACTTCGCATC (SEQ ID NO: 7)

[0124] PNP6-yz-Rev: CTCAACTGCTGTGGCTGTG (SEQ ID NO: 8)

[0125]The PCR product contains a target fragment of a specific size. The length of the PCR fragment verified b...

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Abstract

The invention provides an engineering strain with high yield of FK520 (ascomycin). A gene expression cassette is integrated in a genome of the strain, and the gene expression cassette expresses a protein encoded by a biosynthetic gene of ethyl malonyl coA. The yield of FK520 can be increased substantially by the transformed strain, and the proportion (mass ratio) of the main product FK520 to sideproduct FK523 is increased.

Description

technical field [0001] The invention relates to the field of biotechnology engineering, in particular to a method for constructing an FK520 high-yield engineering strain and a high-yield strain of Streptomyces hygroscopicus. Background technique [0002] FK520, also known as ascomycin, is a 23-membered macrolide antibiotic with immunosuppressive, neuroprotective regeneration and antifungal activities. FK520 is a calcineurin inhibitor, and its mechanism of action is mainly to inhibit the immune activity of T lymphocytes by inhibiting the production of interleukin-2; the inhibitory activity of FK520 on interleukin-2 is stronger than that of cyclosporin A . FK520 can also inhibit the production of IL-8 in neutrophils, and its inhibitory activity on IL-8 is 10 times higher than that of cyclosporine A, while rapamycin does not have the inhibitory activity on IL-8. Therefore, FK520 has great medicinal potential. In addition, pimecrolimus, a new generation of clinically used ato...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12P17/18C12R1/55
Inventor 唐功利姜浩潘海学赵娟李岩
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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