Method for heterologous expression of epothilone

A technology of epothilone and heterologous expression, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of low epothilone expression efficiency and achieve the effect of increasing production

Active Publication Date: 2018-08-28
FUDAN UNIV
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  • Abstract
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Problems solved by technology

[0005] In order to overcome the problem of low epothilone expression efficiency in the prior art

Method used

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  • Method for heterologous expression of epothilone
  • Method for heterologous expression of epothilone
  • Method for heterologous expression of epothilone

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Experimental program
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Effect test

Embodiment 1

[0071] Embodiment 1 Add precursor metabolic pathway pathway gene and tRNA to improve the output of epothilone

[0072] As analyzed previously, the DSM 7029 strain lacks a complete S-methylmalonyl-CoA synthesis pathway, and it lacks the PCC pathway and the mutase-isomerase pathway. Therefore, it is considered to increase the production of epothilone by supplementing the synthesis pathway of S-methylmalonyl-CoA, and the supplementary pathway diagram is shown in figure 1 shown. The method of operation is to first construct a plasmid containing the epo gene cluster and transfer it into the DSM 7029 strain, then construct a plasmid containing different supplementary pathway genes and / tRNA genes, and then transfer it into the above-mentioned DSM7029 strain containing the epo gene cluster to obtain Modified strains.

[0073]The construction process of plasmid 1 (containing the epo gene cluster) is as follows: the plasmids Cosmid 10 and Fosmid3B11 obtained by screening the So0157-2...

Embodiment 2

[0085] Embodiment 2The epo gene cluster is spliced ​​again to add the promoter so as to improve the output of epothilone

[0086] After transcriptome sequencing of the strain MMR1 containing the epothilone gene cluster, it was found that the expression levels of each gene in the epothilone gene cluster were very low ( image 3 ), low-level gene expression may be another key factor restricting the production of epothilone.

[0087] In view of the above situation, in order to improve the expression efficiency of epothilone gene cluster in DSM7029, the six genes of epothilone gene cluster epoA, epoB, epoC, epoD, epoE and epoF were processed as follows by using Bxb1 integrase splicing technology. Figure 4 The resplicing shown. In front of each gene in the epothilone gene cluster, a promoter is added to increase the expression of each gene in the gene cluster. Attempt to increase epothilone production by adding a promoter.

[0088]Specifically, the plasmid pSB1A3 (derived from ...

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Abstract

The invention discloses a method for heterologous expression of epothilone, wherein an epothilone gene cluster is introduced into a host strain while an epothilone precursor synthesis pathway is supplemented, and the introduction of a related tRNA gene and the insertion of a promoter are combined, such that the expression of epothilone can be substantially improved, wherein the yield is increasedby about 4 orders of magnitude, and can reach 8.5 mg/L. The present invention further provides a gene engineering strain capable of heterologous expression of epothilone, and an epothilone productionmethod.

Description

technical field [0001] The invention relates to the field of biological expression, in particular to a method for expressing epothilone heterologously and related genetic engineering strains. Background technique [0002] Epothilones, produced by the fermentation of Sorangium cellulosum, is a new type of antitumor drug similar to paclitaxel with microtubule stabilizing effect. Now there are 5 kinds of epothilone and its derivatives used clinically, among which Patupilone (epothilone B) and KOS-862 (epothilone D) are fermentation products, Ixabepilone and BMS-310705 are epothilone A chemically modified drug of vitamin B, ZK-EPO is a fully chemically synthesized drug. Ixabepilone was approved by the US FDA in October 2007 for the treatment of advanced breast cancer. At present, the main fermentation strain of epothilones is the original strain Somatocys cellulose, and the natural strains mainly produce epothilones A and B. [0003] [Polyangium] brachysporum DSM 7029 (=K481-...

Claims

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Application Information

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IPC IPC(8): C12P17/18C12N1/21C12N1/15C12R1/01C12R1/645
CPCC12N15/52C12P17/181C12P17/18C12R2001/645C12N1/145C12R2001/01C12N1/205
Inventor 丁晓明禹育聪唐标张霖王慧敏卞小莹李越中张友明赵国屏
Owner FUDAN UNIV
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