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Recombinant strain producing terpenoids and construction method thereof, and method for producing terpenoids by fermentation and application thereof

A technology of terpenoids and recombinant strains, which is applied in the field of genetic engineering and microbial fermentation, can solve problems such as hindering industrial application and unfavorable conditions, and achieve the effects of mild conditions, increased yield, and improved fermentation effect

Active Publication Date: 2022-08-02
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on terpenoid synthesis with Yarrowia lipolytica as the host is still mainly concentrated in the cytoplasm, which is not conducive to exploring the potential of Yarrowia lipolytica to produce terpenoids and hinders its industrial application

Method used

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  • Recombinant strain producing terpenoids and construction method thereof, and method for producing terpenoids by fermentation and application thereof

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Embodiment approach

[0029] According to a preferred embodiment of the present invention, the deposit number of the recombinant strain is CGMCC No.24525.

[0030] A second aspect of the present invention provides a method for constructing a recombinant strain, the method comprising: genetically engineering a starting strain so that α-humulene synthase is enhanced in the cytoplasm and peroxisome of the starting strain, respectively activity and flux of mevalonate pathway and acetyl-CoA, or enhance the activity of squalene synthase and flux of mevalonate pathway and acetyl-CoA, respectively; wherein said enhancing α-humulene The mode of synthase activity is to exogenously introduce a gene encoding an α-humulene synthase whose amino acid sequence is shown in SEQ ID NO: 1 in the cytoplasm and peroxisome of the starting strain, and the enhanced squalene The mode of the activity of ene synthase is to exogenously introduce a gene encoding squalene synthase whose amino acid sequence is shown in SEQ ID NO:...

Embodiment 1

[0077] Construction of the original plasmid

[0078] According to Yarrowia lipolytica available on NCBI ( Y.lipolytica ), the nucleotide sequence (genebank accession number AJ306421.1) and hisG tag (genebank accession number AF324729.1) of the orotate nucleoside-5'-phosphate dedredge encoding gene ura, commissioned by Qingke Biotechnology Co., Ltd. The company synthesized and inserted two hisG tag encoding gene sequences into plasmid pUC. Specifically, pUC57 was used as the backbone, and the backbone was recovered after digestion with ECORI and HindIII. The hisG tag encoding gene was cloned in one step using the ClonExpress MultiS One Step Cloning Kit. The plasmid pUC-hisG-hisG was used as the backbone, and the backbone was recovered after digestion with HindIII, and the ura-encoding gene was cloned in one step using the ClonExpressMultiS One Step Cloning Kit. Insert the orotase-5'-phosphate delatease encoding gene into the hisG tag encoding gene sequence, so as to realize t...

Embodiment 2

[0180] Construction of recombinant strains

[0181] will depart strains Yarrowia lipolytica Po1f Δku70 was cultured in YPD liquid medium for 12h until OD600 was 0.8, and the 22 integrated plasmids obtained in Example 1 were transferred into the starting strain by using the Zymogen Frozen EZ YeastTransformation Kit II produced by Zymo Research Corporation for homology. After recombination, competent cells were prepared. After each integrated plasmid was transferred, screening medium was used for screening, and PCR was used to identify correct positive clones.

[0182] It should be noted that the integration plasmids pUC-HUH-intE1-PK-PTA, pUC-HUH-intE2-ACL1-ACL2-AMPD, pUC-intE5-HUH-perCat2, pUC-IntC1-HUH-AAD obtained in Example 1 and pUC-IntC1-HUH-ALDH belong to the recombinant vectors related to the flux enhancement of acetyl-CoA in the cytoplasm of the recombinant strain, and it can be selected for one or more of them; similarly, the integration plasmid pUC-intE3- HUH-PEX10...

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Abstract

The invention relates to the fields of genetic engineering and microbial fermentation, and discloses a recombinant strain producing terpenoids, a construction method thereof, and a method and application for fermenting terpenoids. The recombinant strain is obtained by genetic engineering of the starting strain, and compared with the starting strain, the activity of α-humulene synthase and the flux of mevalonate pathway and acetyl-CoA are respectively enhanced in multiple organelles of the recombinant strain, Or enhance the activity of squalene synthase and the flux of mevalonate pathway and acetyl-CoA, respectively; wherein, the multiple organelles of the recombinant strain include the cytoplasm and peroxisome, and the cytoplasm and peroxisome are respectively Contains a gene encoding α-humulene synthase, or a gene encoding squalene synthase, respectively. The recombinant strain provided by the invention can effectively increase the output of α-humulene or squalene, and has the advantages of raw material regeneration, mild conditions, green production, and is not restricted by time and place.

Description

technical field [0001] The invention relates to the fields of genetic engineering and microbial fermentation, in particular to a recombinant strain producing terpenoids, a construction method thereof, and a method and application for fermenting terpenoids. Background technique [0002] Terpenoids are the largest class of natural secondary metabolites, containing more than 80,000 known molecules, with a high degree of structural diversity and a wide range of applications. Despite this remarkable diversity, all terpenoids are composed of isoprene units (C5). According to the number of C5 units, terpenoids can generally be divided into monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and carotenoids (C40), among which Sesquiterpenes are the most abundant. Alpha-humulene is a molecular formula of C 15 H 24 The monocyclic sesquiterpenes have great potential for application due to their anti-inflammatory, antiallergic and anticancer activities. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N9/10C12N9/12C12N9/04C12N9/02C12N9/90C12N9/88C12N9/80C12N15/31C12N15/53C12N15/54C12N15/55C12N15/60C12N15/61C07K14/39C12P5/00C12R1/645
CPCC12N9/1085C12N9/88C12N9/0006C12N9/1229C12N9/1205C12N9/1025C12N9/1029C12N9/90C12N9/0008C12N9/80C07K14/39C12P5/007C12P5/002C12Y205/01021C12Y402/03104C12Y101/01034C12Y207/04002C12Y205/0101C12Y207/01036C12Y203/0301C12Y401/01033C12Y203/01009C12Y503/03002C12Y102/01003C12Y102/0101C12Y203/01007C12Y305/01028C12Y401/03006C12Y401/02009C12Y203/01008C12Y203/01174C12Y203/03009C12Y203/03003
Inventor 黄和郭琪李珂施天穹
Owner NANJING NORMAL UNIVERSITY
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