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Engineering strain with high yield of FK228 as well as construction and application of engineering strain

An engineering strain, high-yield technology, applied in the direction of bacteria, virus/bacteriophage, microorganism-based methods, etc., can solve the problem of insufficient industrial production

Active Publication Date: 2022-04-29
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the engineering strain E264::R-tdp-dep was obtained by integrating the gene cluster R-tdp-dep into the genome of the bacterial strain Burkholderia thailandensis E264ΔoprABCΔtdpDE5kb that blocked the expression of thailandepsin by transposition, and the yield of FK228 was 94 mg / L, which is still insufficient for industrial production
Based on this, the applicant further searched and found that the complete gene cluster of thailandepsin was knocked out by using Burkholderia high-efficiency homologous recombination system combined with reverse screening technology, and the attB site was added at the position where the thailandepsin gene cluster was knocked out to construct a chassis bacterium E264Δtdp; then through site-specific recombination, the gene cluster R-tdp-dep of FK228 was integrated into the position where the chassis bacteria E264Δtdp originally expressed thailandepsin, and the engineering strain of high-yielding FK228 capable of industrial production and its application have not yet been published. See the report

Method used

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  • Engineering strain with high yield of FK228 as well as construction and application of engineering strain
  • Engineering strain with high yield of FK228 as well as construction and application of engineering strain
  • Engineering strain with high yield of FK228 as well as construction and application of engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of knockout plasmids and helper plasmids for constructing Chassis bacteria E264Δtdp

[0039] For the construction process of plasmid pKY23 and pKP12, see figure 1 .

[0040] The specific steps are as follows: use pBBR1-Rha-ETh1h2e_yi23-kan as the initial plasmid, perform single enzyme digestion on it with Hind III, and perform gel recovery on the plasmid cut into linear fragments; obtain the apramycin resistance gene and the reaction gene by PCR. The sacB gene was screened, and then the two genes were connected in series by tandem PCR; the obtained linear vector fragment and the tandem PCR product were co-transformed into E.coli GB05-dir for line-line recombination, and the obtained recombinant was verified by enzyme digestion. Sequencing, thereby obtaining the Burkholderiathailandensis E264 homologous recombination knockout plasmid, the series of expression plasmids named pKY23, its nucleotide sequence is shown in SEQ ID No.1.

[0041] The p15...

Embodiment 2

[0042] Example 2: Knockout of the tdp gene cluster in thailandepsin biosynthesis in Burkholderiathailandensis E264 For the scheme of knocking out the tdp gene cluster, see figure 2 a.

[0043] The specific steps are: analyze the position of the tdp gene cluster on the chromosome by antiSMASH, and determine that it is located in chromosome I of Burkholderiathailandensis E264 (Accession Number: CP000086.1), and the knockout region is from BTH_I2355 to BTH_I2368, totaling 38.58 kb.

[0044] In order to facilitate the expression of exogenous target biosynthetic gene clusters at the original position of the tdp gene cluster in the later stage, the attB site of the phCi31 site-specific integrase was designed and added.

[0045] The knockout fragment HAUP-tet-PheS-HAUD( ~2.8kb), the knockout fragment contains 160bp homology arms upstream and downstream of the tdp gene cluster, a tetracycline resistance fragment and a PheS reverse selection tag. Wherein, the nucleotide sequence of ...

Embodiment 3

[0075] Example 3: Construction of expression plasmid p15A-oriT-phiC31-kan-R-tdp-dep

[0076] For the construction process of the expression plasmid p15A-oriT-phiC31-kan-R-tdp-dep, see image 3 .

[0077] First, pR6K-oriT-phiC31-kan was used as a template, and oriT-phiC31-kan-1 and oriT-phiC31-kan-2 were used as primers to amplify the fragment oriT-phiC31-kan with p15A-cm-R-tdp on it The homology arms at both ends of the chloramphenicol gene sequence on the -dep plasmid, and the amplified fragments are recovered and purified by gel.

[0078] Electrotransform the purified fragment oriT-phiC31-apra into E.coli GB08-red containing plasmid p15A-cm-R-tdp-dep and induced by 10% L-arabinose. 20 μg / ml apramycin LB plates were placed in a constant temperature incubator at 37°C and cultured overnight. Pick a single colony and digest it with restriction endonuclease PstI (see Figure 4 ), to screen the correct recombinant plasmid p15A-apra-phiC31-R-tdp-dep. The recombinant plasmids w...

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Abstract

The invention discloses an engineering strain E264 [delta] tdp:: R-tdp-dep with high yield of FK228, and the engineering strain E264 [delta] tdp:: R-tdp-dep is obtained by using Burkholderia thailantensis E264 [delta] tdp as an original strain and integrating a combined biosynthetic gene cluster R-tdp-dep on a genome of the original strain through a site specific recombination method. The invention also discloses an application of the engineering strain in fermentation preparation of FK228. Experiments prove that the yield of FK228 produced by the engineering strain E264 [delta] tdp:: R-tdp-dep disclosed by the invention reaches 350mg / L, which is nearly 18 times of the yield of the FK228 produced strain at present. According to the technical scheme, the yield of the FK228 is greatly increased, the fermentation cost is reduced, the fermentation production period is shortened, a foundation is laid for large-scale preparation of the FK228 and development of histone deacetylase inhibitor drugs, and remarkable economic value and social benefit are achieved.

Description

technical field [0001] The present invention relates to an engineering strain producing a histone deacetylase inhibitor and its construction and application, in particular to a high-yield histone deacetylase inhibitor FK228 engineering strain and its construction and application of fermentation to produce FK228. Belongs to the field of biotechnology. Background technique [0002] The use of genetic operating systems to modify bacteria is of great significance for the discovery of new drugs. Currently, the Red homologous recombination technology can be used to precisely modify the genome of bacteria. Previously, the applicant constructed an efficient homologous recombination system in Burkholderia, which contained three homologous recombination operons, among which the operon ETh1h2e_YI23 has a very high homologous recombination efficiency in Burkholderiaglumae PG1, and used the Systematic genome mining in other Burkholderia species revealed a series of novel compounds. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/74C12P17/18C12R1/01
CPCC12N15/52C12N15/74C12P17/188C12N2800/101
Inventor 李瑞娟宫恺王茂芹宋超逸张友明符军李爱英
Owner SHANDONG UNIV
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