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Engineering strain for biosynthesizing mogrol by taking glucose as substrate, construction and application thereof

A mogrosolic alcohol and biosynthesis technology, applied in the biological field, can solve the problems such as the lack of breakthrough progress in the microbial synthesis technology of mogrosolic alcohol, and achieve the effects of low cost, high yield and high purity

Pending Publication Date: 2021-12-07
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

[0003] Through reading a lot of literature, it is found that the microbial synthesis technology of mogrosanol has not made a breakthrough, so it is of great significance to develop an engineering strain and construction method that can industrially produce mogrosanol

Method used

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  • Engineering strain for biosynthesizing mogrol by taking glucose as substrate, construction and application thereof
  • Engineering strain for biosynthesizing mogrol by taking glucose as substrate, construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of Mogrosin-producing Chassis Bacteria

[0030] (1) Construction of Enhanced Squalene Gene Expression Module

[0031] Using the Saccharomyces cerevisiae genome as a template and TEF1-up / down as primers, the promoter PTEF is obtained after PCR amplification; similarly, using the Saccharomyces cerevisiae genome as a template and CYC1t-up / down as primers, the terminator CYC1t is obtained after PCR amplification ; Use the Saccharomyces cerevisiae genome as a template and ERG9-up / down as primers to amplify the ERG9 genome; use the Overlap construction method to assemble the three previous fragments to construct the PTEF1-ERG9-CYCt expression gene cluster module fragment I.

[0032] Using the Saccharomyces cerevisiae genome as a template and TDH3-up / down as primers, the promoter PTDH3 is obtained after PCR amplification; similarly, using the Saccharomyces cerevisiae genome as a template and Ttp1-up / down as primers, amplified to obtain the Ttp1 termina...

Embodiment 2

[0048] Example 2 Construction of Saccharomyces cerevisiae Engineering Bacteria Producing Monk Fruit Alcohol

[0049] 1. Synthesis of Mogrosanol Biosynthesis Module Related Proteins

[0050] Artificially synthesized cucurbitadienol synthase CDS gene sequence, its nucleotide sequence is shown in SEQ ID NO.001, and its nucleotide sequence is shown in SEQ ID NO.002, which is constructed into the pUC19 plasmid vector backbone to obtain the pUC19-CDS plasmid ; artificially synthesized epoxide hydrolase EPH gene sequence, 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 pUC119 plasmid vector backbone to obtain the pUC19-EPH plasmid; artificially synthesized cells P45018D6 gene sequence, the nucleotide sequence is shown in SEQ ID NO.005, the amino acid sequence is shown in SEQ ID NO.006, which is constructed into the pUC19 plasmid vector backbone to obtain the pUC19-CYP87D18 plasmid; artificially ...

Embodiment 3

[0060] Example 3. Fermentation of Saccharomyces cerevisiae producing mogrosan fruit alcohol

[0061] Shake flask fermentation: pick the activated SC-M101 monoclonal on the solid plate, and shake it in YPD liquid medium at 30°C and 250rpm overnight to prepare seed liquid; inoculate the prepared seed liquid into ΔLEU high-density fermentation medium and shake it for 5 days Perform broth measurements. Wherein the fermentation medium formula is as follows:

[0062] Fermentation medium: 25g / L glucose, 15g / L (NH 4 ) 2 SO 4 , 8g / L K 2 HPO 4 , 3g / L MgSO 4 *7H 2 O, 5g / L lysine, 10ml trace elements (containing 15g / L EDTA, 10.2g / L ZnSO 4 *7H 2 O, 0.5g / L MnCl 2 *4H 2 O, 0.86g / LCoCl 2 *6H 2 O, 0.5g / L CuSO 4 , 0.56g / L Na 2 MoO 4 *2H 2 O, 3.84g / L CaCl 2 , 5.12g / L FeSO 4 *7H 2 O), 12ml vitamins (containing 0.05g / L biotin, 0.2g / L p-aminobenzoic acid , 1g / L niacin, 1g / L calcium pantothenate, 1g / L thiamine hydrochloride, 1g / L pyridoxal hydrochloride 1.25g / L inositol). 1L...

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Abstract

The invention discloses an engineering strain for biosynthesizing mogrol by taking glucose as a substrate as well as construction and application of the engineering strain, and belongs to the technical field of biology. A chassis yeast engineering bacterium is constructed by enhancing squalene oxidation module expression, improving an MVA metabolic pathway and knocking out a lanosterol competition pathway, heterologous protein cucurbitadienol synthase, epoxide hydrolase and cell P450 enzyme are expressed on the basis of the chassis bacterium, and the yeast engineering bacterium for biosynthesis of mogrol is constructed. According to the method, mogrol can be synthesized from the beginning, the environment is not polluted, the yield is high, the cost is low, and a basis is provided for industrial production of mogrol.The finally produced mogrol reaches 1.2 g / L, which is the highest level reported at present.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an engineering strain, construction and application for biosynthesizing mogrosanol with glucose as a substrate. Background technique [0002] Mogroside is the aglycon in the synthesis of mogroside, which has the effects of moistening the lungs and relieving cough, clearing away heat and relieving heat, cooling blood and smoothing the intestines, and is a unique economic crop with the same origin of medicine and food in my country; it is used as a commonly used traditional Chinese medicine for the treatment of pharyngitis, whooping cough, Acute and chronic bronchitis, gastrointestinal diseases have remarkable curative effect. In particular, it has a significant improvement effect on the spatial cognitive impairment and learning and memory function decline caused by senile dementia and ischemic dementia. [0003] Through reading a large number of literatures, it is found th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/54C12N15/61C12N15/55C12P33/00C12R1/865
CPCC12N15/81C12N15/52C12N9/90C12N9/14C12N9/0081C12N9/0038C12N9/0071C12N9/1085C12N9/16C12N9/1025C12P33/00C12Y504/99033C12Y303/02003C12Y114/15006C12Y114/14C12Y205/01021C12Y504/99007C12Y301/03047C12Y203/0301
Inventor 赵云现李迪杨志彬胡江林崔金旺田昊博赵凯
Owner 河北维达康生物科技有限公司
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