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Recombinant yarrowia lipolytica for producing scutellarin as well as construction method and application of recombinant yarrowia lipolytica

A technology of Yarrowia lipolytica and construction method, which is applied in the field of molecular biology and bioengineering to achieve the effect of reducing production cost and yield

Active Publication Date: 2022-07-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the heterologous synthesis of scutellarin in Yarrowia lipolytica using synthetic biology methods has not been reported

Method used

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  • Recombinant yarrowia lipolytica for producing scutellarin as well as construction method and application of recombinant yarrowia lipolytica
  • Recombinant yarrowia lipolytica for producing scutellarin as well as construction method and application of recombinant yarrowia lipolytica
  • Recombinant yarrowia lipolytica for producing scutellarin as well as construction method and application of recombinant yarrowia lipolytica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Construction of recombinant plasmids

[0113] (A), G1-KU80, G2-IntC-2, G3-IntC-3, G4-IntD-4, G5-IntE-1, G6-IntE-3, G8-ARO10, G10-D17 knockout vector construction

[0114] Using the PgRNA-YL vector as a template, the upstream and downstream fragments of G1-KU80 were amplified with primers pgRNA-ZHONG-5F / G1-3R and pgRNA-ZHONG-3R / G1-5F, and primers pgRNA-ZHONG-5F / G2- 3R and primer pgRNA-ZHONG-3R / G2-5F were amplified to obtain upstream and downstream fragments of G2-IntC-2, and G3 was amplified with primer pgRNA-ZHONG-5F / G3-3R and primer pgRNA-ZHONG-3R / G3-5F. -IntC-3 upstream and downstream fragments, use primer pgRNA-ZHONG-5F / G4-3R and primer pgRNA-ZHONG-3R / G4-5F to amplify the upstream and downstream fragments of G4-IntD-4, use primer pgRNA-ZHONG-5F / G5-3R and primers pgRNA-ZHONG-3R / G5-5F were amplified to obtain upstream and downstream fragments of G5-IntE-1, which were amplified with primers pgRNA-ZHONG-5F / G6-3R and pgRNA-ZHONG-3R / G6-5F The upstream and dow...

Embodiment 2

[0142] Example 2. Construction of recombinant strains

[0143] (1), the construction of recombinant strain 1

[0144] Use primer YL-p1-F / R to linearize the integration vector P1-4CL-CHS to obtain 4CL and CHS gene expression cassettes, and use primer YL-p2-F / R to linearize the integration vector P2-FSII to obtain FSII Gene expression cassette, use primer YL-p3-F / R to linearize the integration vector P3-SbarCYP82D4-ATR2 to obtain CYP82D4 gene and ATR2 gene expression cassette, use primer YL-p4-F / R to integrate vector P4-EbF7GAT- EbUDPGDH was linearized to obtain F7GAT gene and UDPGDH gene expression cassettes. The integration vector P6-EbPAL-EbC4H was linearized with primers YL-p6-F / R to obtain PAL gene and C4H gene expression cassettes.

[0145] The 4CL and CHS gene expression cassettes and the knockout vector G1-KU80 were integrated into the Yarrowia lipolytica W29Δku70 strain by the method of lithium acetate, and then the FSII gene expression cassette and the knockout vector...

Embodiment 3

[0152] Example 3. Application of Yarrowia lipolytica genetically engineered bacteria producing scutellarin

[0153] (1) Cultivation of engineering bacteria and product extraction

[0154] Recombinant bacteria 1-4 in Example 2 were used to produce scutellarin. The specific method is as follows: the recombinant bacteria are activated and cultured in YNB liquid medium at 30° C. and 220 rpm for 48 hours to obtain seed liquid. The seed liquid was inoculated into 30 ml of YNB fermentation medium at an inoculation amount of 1%, and cultured with shaking at 30° C. and 220 rpm for 4 days. After fermentation, 500 microliters of fermentation broth was taken and 500 microliters of methanol was thoroughly mixed, sonicated for 30 minutes, and centrifuged at 12,000 rpm for 20 minutes. Take 200 μl for product detection.

[0155] (2), HPLC, LC-MS detection conditions

[0156] HPLC analysis: Instrument: Shimadzu high performance liquid chromatograph 1200; Chromatographic column: Kinetex H15...

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Abstract

The invention discloses recombinant yarrowia lipolytica for producing scutellarin as well as a construction method and application of the recombinant yarrowia lipolytica. The construction method comprises the following steps: firstly, realizing de novo production of the scutellarin in the yarrowia lipolytica by heterologous expression of nine key enzymes synthesized by a scutellarin pathway in the yarrowia lipolytica, and further enabling the yield of the scutellarin to reach 94.79 mg / L under a shake flask condition through optimization and transformation; after fed-batch fermentation, the yield of scutellarin can reach 346 mg / L, the proportion of scutellarin reaches 80% or above, and the proportion of by-products is small. The method provided by the invention lays a foundation for efficient synthesis of scutellarin by artificial cells.

Description

technical field [0001] The invention belongs to the fields of molecular biology and bioengineering, and particularly relates to recombinant Yarrowia lipolytica for heterologous synthesis of scutellarin and its application. Background technique [0002] As one of the bioactive components of breviscapine flavonoid glycosides, breviscapine can be used not only for the treatment of cardiovascular and cerebrovascular diseases, but also for several other chronic diseases, such as diabetic complications, stroke, kidney disease and non-alcoholic fatty liver disease. The main source of scutellarin is still extracted from plants, which greatly depletes the wild resources of this species. Chemical synthesis is overshadowed by the use of expensive starting materials and tedious synthetic procedures. Therefore, the use of synthetic biology technology to synthesize breviscapine is an effective means to solve the shortage of breviscapine resources. [0003] [0004] In recent years, m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N15/53C12N15/52C12N15/54C12N15/81C12N1/19C12P19/60C12R1/645
CPCC12N9/88C12N9/0071C12N9/93C12N9/1037C12N9/1051C12N9/0006C12N9/0042C12N15/52C12N15/815C12P19/60C12Y403/01024C12Y114/14C12Y602/01012C12Y203/01074C12Y114/11022C12Y204/01081C12Y101/01022C12Y106/02004
Inventor 江会锋王益娜刘晓楠陈碧环刘伟卢丽娜张广辉杨生超
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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