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A recombinant Yarrowia lipolytica producing α-humulene and its construction method and application

A technology of Yarrowia lipolytica and Yarrowia lipolytica, applied in the direction of microorganism-based methods, biochemical equipment and methods, applications, etc., to achieve the effects of simple operation, high-efficiency synthesis, and increased yield

Active Publication Date: 2022-06-14
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Yarrowia lipolytica is an unconventional safe oleaginous yeast certified by the FDA. Its advantages are sufficient intracellular acetyl CoA / cofactor supply and cell tolerance Well, the generated oil can also wrap lipophilic terpenoids to enhance the storage capacity of terpenoids. In addition, Gibson, Gateway, BioBricks, one-step assembly methods in vivo have been established in Yarrowia lipolytica; and genome manipulation tools include TALEN, CRISPR / Cas9, CRISPRa, CRISPRi, Cpf1, base editor, Cre / Loxp, URA / TRP Blaster, and metabolic engineering techniques include push-pull strategy, multi-copy integration strategy, promoter / terminator engineering, regionalization strategy, random integration / PiggyBac transposition screening system, computer-aided design of genome-scale metabolic network and other DNA assembly tools; so far, there is no report on the heterologous high-production of α-humulene in Yarrowia lipolytica

Method used

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  • A recombinant Yarrowia lipolytica producing α-humulene and its construction method and application
  • A recombinant Yarrowia lipolytica producing α-humulene and its construction method and application
  • A recombinant Yarrowia lipolytica producing α-humulene and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0044] 1. Amplification of genetic elements and preparation of target plasmids

[0045] 1. Preparation of target gene

[0046] According to the nucleotide sequence (KT893310) of the α-humulene synthase coding gene ACHS2 provided by NCBI, after specific codon optimization, we commissioned Qingke Biological Co., Ltd. to synthesize the optimized α-humulene synthase code The gene ACHS2 was inserted into the plasmid pUC57 to obtain the plasmid pUC-ACHS2. The optimized nucleotide sequence of ACHS2 is shown in SEQ ID NO.1.

[0047] According to the nucleotide sequence (M37309.1) of the 3(B)-isopropylmalate dehydrogenase coding gene leu in Yarrowia lipolytica provided on NCB1, Qingke Biotechnology Co., Ltd. was entrusted to synthesize leu, and 3(B) )-Isopropylmalate dehydrogenase coding gene was inserted into the plasmid PUC to obtain the plasmid PUC-leu. According to the nucleotide sequence (genebank accession number AJ306421.1) and hisG tag (genebank accession number AJ306421.1) ...

Embodiment 2

[0081] Construction of recombinant bacteria

[0082] 1. Construction of Recombinant Bacteria 1

[0083] 1. The plasmid PUC-leu-A08-ACHS2 containing the ACHS2 gene expression cassette was introduced into Yarrowia lipolyticaPolf-Δku70, and the ACHS2 expression cassette was integrated into the A08 site of the genome to obtain recombinant bacterium 1. The specific method is as follows: (1) Yarrowia lipolytica Polf-Δku70 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose), and the OD 600 When growing to between 0.8-1.0, prepare competent cells (kit: ZymogenFrozen EZ Yeast Transformation Kit II, manufacturer: Zymo Research Corporation). (2) Use the Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation to transform PUC-leu-A08-ACHS2 into Yarrowia lipolytica Polf-Δku70 for homologous recombination. (3) The screening medium SD-Leu was used to screen, and the positive clone identified by PCR was named recombinant ba...

Embodiment 3

[0087] Application of recombinant bacteria 1 and 2 in the production of α-humulene

[0088] 1. Cultivation of engineering bacteria and product extraction

[0089] The recombinant bacteria 1 and 2 in Example 2 were used to produce α-humulene respectively. The specific method is as follows: activate the recombinant bacteria, culture in YPD liquid medium at 30° C. and 220 rpm for 16 hours to obtain seed liquid. The seed solution was inoculated in 50ml of fermentation medium with an inoculum of 1% by volume, and cultured with shaking at 30°C and 220rpm for 1 day, then adding n-dodecane with a volume of 25% of the fermentation broth, and continuing to shake for 3 days. After the fermentation, the fermentation broth was transferred to a 50ml centrifuge tube, centrifuged at 5000rpm for 15min, and the upper organic phase was collected for later use.

[0090] Wherein the fermentation medium contains 60g / L glucose, 10g / L yeast powder and 20g / L trypsin.

[0091] 2. Qualitative and qua...

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Abstract

The invention discloses a recombinant Yarrowia lipolytica that produces α-humulene and its construction method and application. The recombinant Yarrowia lipolytica is inserted into the Yarrowia lipolytica genome to optimize the α- Hupulene synthase coding gene ACHS2 and tHMGR coding gene were obtained. The recombinant Yarrowia lipolytica of the present invention can efficiently synthesize α-humulene, the construction method is efficient, and the operation is simple. In addition, the method for collecting the product provided by the present invention adopts the method of extracting while fermenting, and the produced α-humulene is extracted in a timely manner. The extraction of humulene into the organic phase relieves the pressure of dissolving α-humulene in the cell, effectively increases the output of α-humulene, and lays the foundation for the synthesis of α-humulene by artificial cells.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a recombinant Yarrowia lipolytica for producing α-humulene, a construction method and application thereof. Background technique [0002] Terpenoids, also known as isoprenoids, are a class of compounds ubiquitous in the plant kingdom, including more than 50,000 molecules with various molecular sizes, structures, and biological functions. According to the number of isoprene units in the molecule, terpenes can be divided into: hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesquiterpenes, triterpenes, tetraterpenes, polyterpenes, etc. Terpenoids play an important role in life activities. In addition, terpenoids have important application value in medicine, energy, food and other fields. Therefore, it is very necessary to excavate these terpenoids with a large number and various structures in nature and realize their wide application value. [0003] The large-scale...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/60C12N15/53C12N15/81C12P5/00C12R1/645
CPCC12N9/88C12N9/0006C12N15/815C12P5/007C12P5/002C12Y402/03104C12Y101/01034
Inventor 黄和郭琪施天穹孙小曼彭倩倩
Owner NANJING NORMAL UNIVERSITY
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