Saccharomyces cerevisiae construction method for efficiently synthesizing alpha-amyrin

A technology of balsamol and Saccharomyces cerevisiae, which is applied in the field of bioengineering, can solve problems such as low extraction efficiency, serious pollution, and limited yield, and achieve the effects of improving catalytic activity, reducing damage, and increasing production

Pending Publication Date: 2020-04-10
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditional chemical synthesis and plant source extraction have

Method used

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  • Saccharomyces cerevisiae construction method for efficiently synthesizing alpha-amyrin
  • Saccharomyces cerevisiae construction method for efficiently synthesizing alpha-amyrin
  • Saccharomyces cerevisiae construction method for efficiently synthesizing alpha-amyrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Screening of MdOSC1 mutants

[0027] In order to perform molecular docking on the wild-type α-amyresinol synthase MdOSC1 from apple, this study used the reported human lanosterol synthase (PDB: 1W6K ) was used as a homologous protein template, and the 3D structure of MdOSC1 was homologously modeled by the online tool SWISS-MODEL (http: / / www.swissmodel.expasy.org). At the same time, use the online tool Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ) to predict the 3D structure of the substrate (3S)-2,3-oxide squalene, and download MdOSC1 and (3S)-2,3 respectively - The structure file of squalene oxide, using AutoDock 1.5.6 for molecular docking simulation, respectively importing the structure files of MdOSC1 and (3S)-2,3-oxide squalene, where (3S)-2,3-oxide squalene ( PubChem CID: 5459811) as substrate, operate according to the following procedures:

[0028] Edit>Charges>Add Kollman Charges

[0029] Edit>Hydrogens>Add(Hydrogens)>Polar Only>OK

[0030] WithBondO...

Embodiment 2

[0040] Example 2 Construction of mutant MdOSC1-1 high expression plasmid

[0041] The pYYG vector is a truncated vector based on the pESC plasmid. The plasmid is minimized by removing non-essential sequences in the plasmid. The pESC-Trp-MdOSC1-1 plasmid is used as a template, and the upstream and downstream primers are respectively introduced into the BamHI and XhoI restriction sites. , PCR amplification of the MdOSC1-1 gene, the PCR reaction system is shown in Table 2, and the PCR reaction conditions are specifically set according to the actual conditions such as the primer composition and the size of the amplified fragment.

[0042] Table 2 PCR reaction system

[0043]

[0044] PCR reaction conditions: from the second step to the fourth step, repeat 30 cycles.

[0045]

[0046] Construction of pYYG-Trp-MdOSC1-1 plasmid:

[0047] The PCR product of MdOSC1-1 and the pYYG-Trp vector were digested with BamHI and XhoI restriction enzymes, and the target fragment was inser...

Embodiment 3

[0055] Example 3 Co-expression of key genes and diacylglycerol acyltransferase (DGA1) genes in the MVA pathway

[0056] The key gene expression cassettes of metabolic pathways were assembled using the same method as the construction of plasmid gene expression cassettes P TDH3 -tHMG1-T HMG1 , while using the pESC-His plasmid as a template, PCR amplifies the His selection gene expression cassette P TEF1 -HIS3-T ADH1 . Using yeast genomic DNA as a template, the homology arms of rDNA loci were amplified by PCR. The primers used to construct the genome assembly strain AM1 are shown in Table 5, and the underlined primers indicate overlapping regions.

[0057] Table 5 Gene expression cassettes ERG20, ERG9, ERG1 genome primers

[0058]

[0059]

[0060] Assembly of gene expression cassette P by PCR DGA1 -DGA1-T DGA1 At the same time, using the pESC-Leu plasmid as a template, PCR amplified the Leu screening gene expression cassette P TEF1 -HIS3-T ADH1 . Using yeast geno...

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Abstract

The invention provides a method for constructing Saccharomyces cerevisiae for efficiently synthesizing alpha-amyrin, which belongs to the field of bioengineering. The method comprises the following steps: carrying out semi-rational design on alpha-amyrin synthase derived from Malus domestica, and carrying out saturated mutation and iterative mutation on key amino acid residue sites to obtain a series of mutants including an MdOSC1 mutant with the highest catalytic activity; then performing overexpression on key genes in an alpha-amyrin synthesis route, and meanwhile, performing overexpressionon diacylglycerol acyltransferase (DGA1) so that the storage capacity of alpha-amyrin in saccharomyces cerevisiae cells is improved. The yield of the saccharomyces cerevisiae engineering strain capable of producing alpha-amyrin at high yield constructed by the method reaches 213.79 mg/L, efficient production of alpha-amyrin in saccharomyces cerevisiae is realized, and the engineering strain has important significance in production.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a construction method and application of Saccharomyces cerevisiae for efficiently synthesizing α-amyresinol. Background technique [0002] α-Arysinol is an important ursane-type pentacyclic triterpenoid with various common biological activities, including anti-tumor, anti-inflammatory, anti-oxidation, immune enhancement and liver protection, and has the strongest anti-proliferative active. As a plant secondary metabolite, α-amyresinol is widely distributed in leaves, fruits and rhizomes of plants, but the accumulation amount is small. Traditional chemical synthesis and plant source extraction have low efficiency, limited yield, long cycle and serious pollution. With the rapid development of synthetic biology and metabolic engineering, the construction of recombinant microbial engineering bacteria with heterologous gene integration can be used as a solution for the prod...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/90C12N15/61C12N15/53C12N15/54C12P33/00C12R1/865
CPCC12N9/0006C12N9/1029C12N9/90C12N15/52C12N15/81C12N15/905C12P33/00C12Y101/01088C12Y203/0102C12Y504/9904
Inventor 王颖余源常鹏程刘皓然李春
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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