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Engineering bacterium for efficiently producing patchouli alcohol, and construction method and application of engineering bacterium

A technology of patchouli alcohol and engineering bacteria, applied in the field of bioengineering, can solve the problems of lack of patchouli alcohol sustainability and large-scale production, and achieve the effect of improving genetic stability and simple operation

Active Publication Date: 2021-10-26
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microbial production thus offers an attractive approach for the sustainable and large-scale production of patchouli alcohol, which currently does not exist for sustainable and large-scale production of patchouli alcohol.

Method used

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  • Engineering bacterium for efficiently producing patchouli alcohol, and construction method and application of engineering bacterium
  • Engineering bacterium for efficiently producing patchouli alcohol, and construction method and application of engineering bacterium
  • Engineering bacterium for efficiently producing patchouli alcohol, and construction method and application of engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Amplification of genetic elements and preparation of target plasmids

[0043] 1. Preparation of target gene

[0044] According to the nucleotide sequence of the patchouli alcohol synthase coding gene PS1 provided by NCBI (number 589912511, 1659bp on NCBI), after the inventor’s specific codon optimization, Nanjing Qingke Biotechnology Co., Ltd. was entrusted to synthesize the optimized patchouli alcohol synthase Huoyangol synthase encoding gene PS1 (about 79% homology with the original sequence) was inserted into plasmid pUC57 to obtain plasmid pUC57-PS1. The optimized nucleotide sequence of PS1 is shown in SEQ ID NO.1.

[0045] According to the nucleotide sequence (M37309.1) of the 3(B)-isopropylmalate dehydrogenase coding gene leu in Yarrowia lipolytica provided on NCB1, Nanjing Qingke Biotechnology Co., Ltd. was entrusted to synthesize leu, and 3( B) - The gene encoding isopropylmalate dehydrogenase was inserted into plasmid pUC to obtain plasmid pUC-leu.

[004...

Embodiment 2

[0080] Construction of recombinant bacteria

[0081] 1. Construction of Recombinant Bacteria 1

[0082] 1. The plasmid pUC-leu-A08-PS1 (sequence shown in SEQ ID NO.6) containing the optimized PS1 gene expression cassette was introduced into Yarrowia lipolytica Polf-Δku70, and the PS1 expression cassette was integrated into the A08 site of the genome to obtain Recombinant bacteria1. 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 0.8-1.0, prepare competent cells (kit: Zymogen Frozen EZ Yeast Transformation Kit II, manufacturer: ZymoResearch Corporation). (2) Transform pUC-leu-A08-PS1 into Yarrowia lipolytica Polf-Δku70 using the Zymogen Frozen EZYeast Transformation Kit II of Zymo Research Corporation for homologous recombination. (3) The screening medium SD-Leu was used to screen, and the positive clone identified by PCR ...

Embodiment 3

[0087] Application of Recombinant Bacteria 1 and 2 in the Production of Patchouli Alcohol

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

[0089] The recombinant bacteria 1 and 2 in Example 2 were used to produce patchouli alcohol respectively. The specific method is as follows: activate the recombinant bacteria, culture in YPD liquid medium at 30° C. and 200 rpm for 15 hours to obtain seed liquid. The seed solution was inoculated in 50ml fermentation medium with an inoculation amount of 1% volume ratio, and cultured with shaking at 30°C and 200rpm for 1 day, then added n-dodecane with 25% volume of the fermentation broth, and continued shaking culture 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...

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Abstract

The invention discloses an engineering bacterium for efficiently producing patchouli alcohol, and a construction method and application of the engineering bacterium; the engineering bacterium is obtained by inserting optimized patchouli alcohol synthase coding genes PS1 and tHMGR into a genome of yarrowia lipolytica serving as an original strain; the optimized patchouli alcohol synthase coding gene PS1 is as shown in SEQ ID NO. 1; and the tHMGR coding gene is as shown in SEQ ID NO. 2. The engineering bacterium disclosed by the invention can efficiently synthesize the patchouli alcohol; the construction method is efficient and easy to operate; in addition, the method for collecting the product provided by the invention adopts a method of extracting while fermenting; the produced patchouli alcohol is extracted into an organic phase in time; the pressure of intracellular dissolution of the patchouli alcohol is relieved; the yield of the patchouli alcohol is effectively increased; and a foundation is laid for synthesizing the patchouli alcohol by artificial cells.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an engineering bacterium for efficiently producing patchouli alcohol, a construction method and application thereof. Background technique [0002] Plant-derived natural products refer to a class of secondary metabolites with biological activity in plants. The diversity of plant species and ecological environment has created the rich diversity of the structure and performance of plant natural products. Therefore, the types and quantities of plant natural products are very large. The currently known types mainly include terpenes, alkaloids, and steroids. , flavonoids and phenols. Among them, terpenoids are a large class of substances with the largest number and widest distribution in plant natural products, which are a general term for a series of compounds with isoprene as a structural unit. Most terpenoids have pharmacological and biological activities, and can also be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/02C12R1/645
CPCC12N9/88C12N15/815C12Y402/0307C12P7/02
Inventor 黄和彭倩倩沈琦杨彩玲施天穹
Owner NANJING NORMAL UNIVERSITY
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