Yarrowia lipolytica engineering strain for biologically synthesizing glycyrrhetinic acid by taking glucose as substrate as well as construction and application thereof

A technology of Yarrowia lipolytica and glycyrrhetinic acid, which is applied in the biological field, can solve the problems of endangering human safety, long routes, and staying, and achieve the effect of being friendly to the human body and the environment

Pending Publication Date: 2022-01-14
河北维达康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the synthesis method of glycyrrhetinic acid is mainly through the solvent extraction method, which obtains glycyrrhizic acid powder through many steps such as extraction, separation, precipitation, and drying of licorice, and then obtains glycyrrhetinic acid through extraction and crystallization. The synthesis steps are many and the route is long. , high cost, low yield, and low purity are not conducive to the industrial production of glycyrrhetinic acid. At the same time, dangerous reagents need to be used in the operation process, which seriously endangers human safety.
There are also methods of using Saccharomyces cerevisiae as a host to produce glycyrrhetinic acid using synthetic biology methods, but they all remain at the stage of synthesizing glycyrrhetinic acid with glycyrrhizic acid as a precursor

Method used

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  • Yarrowia lipolytica engineering strain for biologically synthesizing glycyrrhetinic acid by taking glucose as substrate as well as construction and application thereof
  • Yarrowia lipolytica engineering strain for biologically synthesizing glycyrrhetinic acid by taking glucose as substrate as well as construction and application thereof
  • Yarrowia lipolytica engineering strain for biologically synthesizing glycyrrhetinic acid by taking glucose as substrate as well as construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Construction of Glycyrrhetinic Acid-Producing Yarrowia lipolytica Engineering Bacteria

[0031] (1) Synthesis of the coding sequence of glycyrrhetinic acid synthesis-related protein

[0032] Artificially synthesized β-amyresinol synthase bAS, whose nucleotide sequence is shown in SEQ ID NO:001, and whose amino acid sequence is shown in SEQ ID NO:002, which was constructed as the pUC19 plasmid vector backbone to obtain the pUC19-bAS plasmid; artificially synthesized NADPH -Cytochrome P450 reductase gene CPR, its nucleotide sequence is shown in SEQ ID NO:003, and its amino acid sequence is shown in SEQ ID NO:004, which is constructed into the pUC19 vector backbone to obtain the pUC19-CRP plasmid; artificially synthesized cytochrome P450 The oxidase CYP88D6 gene, whose nucleotide sequence is shown in SEQ ID NO:005, and its amino acid sequence is shown in SEQ ID NO:006, was constructed into the pUC19 vector backbone to obtain the pUC19-CYP88D6 plasmid; artifici...

Embodiment 2

[0042] Embodiment 2 produces the fermentation and detection of glycyrrhetinic acid

[0043] (1) Shake flask fermentation: YPD medium is used for the cultivation and fermentation of Yarrowia lipolytica. For the single fermentation of shake flask, first pick a single clone of GA001 strain from the plate and inoculate it into 10ml of YPD medium for seeding The activated seed solution was then transferred to a 250ml shake flask, loaded with 30mL of YPD medium, and cultured in a shaker at 30°C at 220rpm for 4 days. All shake flask fermentation experiments were set up with 3 parallel experimental groups. After the fermentation is finished, the product detection is carried out in the manner shown in Example 2 (2);

[0044] (2) The cells were centrifuged at 6000rpm for 10 minutes to separate the supernatant and bacteria, and the bacteria were added with 4ml of 1-butanol and quartz sand and vortexed for 20 minutes to extract intracellular products. After evaporating the obtained bu...

Embodiment 3

[0047] Example 3 Weakening lanosterol synthase and enhancing the expression of squalene synthase and squalene cyclooxygenase to improve the expression of licorice Acid production

[0048] (1) Construction of squalene synthase (ERG9) and squalene cyclooxygenase (ERG1) expression modules

[0049] Php4d-ERG9-xpr2 expression module construction: using hp4d-up / down and xpr2t-up / down as primers and pINA1269 plasmid as a template, the Php4d promoter and xpr2t terminator were respectively amplified; using ERG9-F / R as primers, The Yarrowia lipolytica genome was used as a template to amplify the squalene synthase ERG9 gene, and the Overlap method was used to construct the Php4d-ERG9-xpr2 expression module.

[0050] Construction of pTEFin-ERG1-lip2t expression module: using pTEFin-up / down and lip2t-up / down as primers, pMT015 plasmid as template, amplified to obtain PTEFin promoter and lip2t terminator; using ERG1-F / R as primers, The Yarrowia lipolytica genome was used as a template ...

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Abstract

The invention discloses a yarrowia lipolytica engineering strain for biologically synthesizing glycyrrhetinic acid by taking glucose as a substrate as well as construction and application thereof, and belongs to the technical field of biology. According to the engineering strain, a beta-amyrin synthase expression module, a 11 / 30 site-oxidized beta-amyrin synthase expression module, a cell P450 oxidase expression module and a cytochrome P450 reductase expression module are integrated on a genome; the construction method comprises the following steps: constructing to obtain glycyrrhetinic acid biosynthesis module plasmids: beta-amyrin synthase bAS, cytochrome P450 oxidase 88D6CYP88D6, 11 / 30-site-oxidized beta-amyrin synthase CYP725A4 and cytochrome P450 reductase CPR, integrating the glycyrrhetinic acid biosynthesis module onto a yarrowia lipolytica tool plasmid, and linearizing the plasmids integrated with the glycyrrhetinic acid biosynthesis module, and introducing the linearized plasmids into yarrowia lipolytica to obtain the yarrowia lipolytica engineering bacteria of which the genome is integrated with the glycyrrhetinic acid biosynthesis module. The yield of glycyrrhetinic acid produced by the engineering strain is remarkably improved and reaches 7g / L to the maximum.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to an engineering strain of Yarrowia lipolytica which uses glucose as a substrate to biosynthesize glycyrrhetinic acid, its construction and its application. Background technique [0002] Glycyrrhiza licorice is a kind of traditional Chinese medicine for invigorating qi blindly, and it is the most widely used traditional Chinese medicine in clinical practice. Licorice is sweet in taste and flat in nature, enters the heart, spleen, lung, and stomach meridians, and has the functions of invigorating the spleen and nourishing qi, moistening the lungs and relieving cough, clearing away heat and detoxification, relieving pain, relieving medicinal properties, etc. Glycyrrhetinic acid and glycyrrhizic acid are the main drugs in licorice Glycyrrhetinic acid is the precursor of glycyrrhizic acid biosynthesis, and has similar pharmacophysiological activities to glycyrrhizic aci...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/90C12N15/61C12N15/53C12P33/00C12R1/645
CPCC12N15/52C12N9/90C12N9/0073C12N9/0042C12N15/815C12N15/905C12P33/00C12Y504/99039C12Y114/13134C12Y106/02004
Inventor 赵云现李迪杨志彬崔金旺田昊博胡江林展全乐
Owner 河北维达康生物科技有限公司
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