Engineering strain for heterologous expression of histone deacetylase inhibitor FK228 as well as construction and application of engineering strain

A sirtuin, FK228 technology, applied in the field of biosynthesis, can solve the problems of lack of engineering strains, difficult chemical synthesis, limited yield, etc.

Pending Publication Date: 2021-11-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the yield of the wild strain Chromobacterium violaceum No.968 producing FK228 is relatively limited (19mg / L), and its chemical synthesis has also been proved to be very difficult. Engineering strain of acetylase inhibitor FK228

Method used

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  • Engineering strain for heterologous expression of histone deacetylase inhibitor FK228 as well as construction and application of engineering strain
  • Engineering strain for heterologous expression of histone deacetylase inhibitor FK228 as well as construction and application of engineering strain
  • Engineering strain for heterologous expression of histone deacetylase inhibitor FK228 as well as construction and application of engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of plasmid pBR322-amp-ccdB-depD-depE

[0049] For the construction process of plasmid pBR322-amp-ccdB-depD-depE, see figure 1 .

[0050] The specific steps are: according to the DNA sequence of the FK228 biosynthetic gene cluster in NCBI, the genes depD and depE are divided into three sections for synthesis. The three DNA sequences are respectively named PartI, PartII, and PartIII, and each of the three fragments has homology arms, which can be assembled with the vector. Using pBR322-amp-ccdB-neo-rpsL as a template, the pBR322-amp-ccdB fragment was amplified with primers BR322-ampccdB-DE-1 and BR322-ampccdB-DE-2 to obtain the PCR product pBR322-amp-ccdBPCR. The two ends of the pBR322-amp-ccdB PCR have the homology arms of PartI and PartIII respectively, and pBR322-amp-ccdBCR is recovered by gel cutting. Incubate PartI, PartII, PartIII and pBR322-amp-ccdB PCR with T4 DNA polymerase in vitro, and then electrotransfer to E. coliGBdir-gyrA462 indu...

Embodiment 2

[0064] Example 2: Construction of plasmid p15A-apra-tnpA-R-tdp-dep

[0065] For the construction process of plasmid p15A-apra-tnpA-R-tdp-dep, see image 3.

[0066] (1) Construction of plasmid p15A-cm-tdp-delDE-km-ccdB

[0067] Using pR6K-km-ccdB as a template, using kmccdB-1 and kmccdB-2 as primers to amplify the km-ccdB fragment, the two ends of the fragment have homology arms at both ends of the tdpDE gene, and the obtained fragment km-ccdB PCR was carried out After the gel was recovered, the purified km-ccdB PCR fragment was electrotransferred into E.coliGBred-gyrA462 carrying the plasmid p15A-cm-tdp and induced by 10% L-arabinose. Place on LB plates with 30 μg / ml kanamycin in a constant temperature incubator at 37°C and culture overnight. Pick a single colony, and carry out double digestion with restriction endonucleases PstI and AscI (see Figure 4 ), to screen the correct recombinant plasmid p15A-cm-tdp-delDE-km-ccdB. The recombinant plasmids with correct restricti...

Embodiment 3

[0121] Embodiment 3: Construction of the engineering strain E264 of the present invention::R-tdp-dep

[0122] The expression plasmid p15A-apra-tnpA-R-tdp-dep was electrotransformed into E.coli WM3064, and then combined with Burkholderia thailandensis E264ΔoprABCΔtdpDE5kb for transfer, and finally the combined biosynthetic gene cluster R-tdp-dep was integrated into the host chromosome to obtain The engineered strain E264::R-tdp-dep capable of producing FK228.

[0123] Conjugative transfer step: First, electrotransfer the plasmid p15A-apra-tnpA-R-tdp-dep into E.coliWM3064, pick a single clone, carry out enzyme digestion identification and screen the correct clone; the plasmid containing p15A-apra-tnpA-R-tdp-dep E.coliWM3064 was streaked on LB plate with 20μg / ml apramycin and 1mM DAP, 37℃, cultured upside down overnight; Burkholderia thailandensis E264ΔoprABCΔtdpDE5kb was streaked on LB plate, 37℃, cultured upside down overnight; picked overnight The cultured Burkholderia thaila...

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Abstract

The invention discloses an engineering strain for heterologous expression of a histone deacetylase inhibitor FK228. The engineering strain is named as an engineering strain E264:: R-tdp-dep, and the engineering strain is obtained by using Burkholderia thailanensis E264 [delta] oprABC [delta] tdpDE5kb as an original strain and integrating a combined biosynthetic gene cluster R-tdp-dep on a genome of an original strain through a transposition method. The invention also discloses an application of the engineering strain in preparation of the histone deacetylase inhibitor FK228. Experiments prove that the yield of FK228 produced by the engineering strain E264:: R-tdp-dep reaches 94 mg/L and is nearly 5 times that of a current FK228 industrial production strain, 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 and development of histone deacetylase inhibitor drugs, The engineering strain has obvious economic value and social benefit.

Description

technical field [0001] The present invention relates to an engineering strain for producing histone deacetylase inhibitor and its construction and application, in particular to an engineering strain for heterologously expressing histone deacetylase inhibitor FK228 and its construction and fermentative production of FK228 The application belongs to the technical field of biosynthesis. Background technique [0002] Histone deacetylase inhibitors (histone deacetylases inhibitor, HDACi) can prevent histone deacetylases (histone deacetylases, HDACs) from removing the acetyl group on the N-terminal lysine residues of histones, so that chromatin maintains a A more open transcriptionally active state that can regulate the expression of silent tumor suppressor genes. FK228, also known as Romidepsin or Depsipeptide, was originally isolated from Chromobacterium violaceum in Japanese soil samples. As a highly efficient and selective histone deacetylase inhibitor, FK228 has very good a...

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
CPCC12N15/52C12N15/74C12P17/188
Inventor 李瑞娟宋超逸张友明李爱英王茂芹王宗杰宫恺
Owner SHANDONG UNIV
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