Method for synthesizing S-adenosylmethionine from DL-methionine by modifying saccharomyces cerevisiae based on CRISPR technology

A technology of adenosylmethionine and Saccharomyces cerevisiae, applied in the direction of microorganism-based methods, other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve the problems of less development and research, and achieve the effect of reducing costs and improving efficiency

Inactive Publication Date: 2021-02-05
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Less research has been done on tool development for polyploid industrial ye

Method used

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  • Method for synthesizing S-adenosylmethionine from DL-methionine by modifying saccharomyces cerevisiae based on CRISPR technology
  • Method for synthesizing S-adenosylmethionine from DL-methionine by modifying saccharomyces cerevisiae based on CRISPR technology
  • Method for synthesizing S-adenosylmethionine from DL-methionine by modifying saccharomyces cerevisiae based on CRISPR technology

Examples

Experimental program
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Effect test

Example Embodiment

[0028] Example 1 Selection of CRISPR / Cas9 System in Industrial Saccharomyces cerevisiae

[0029] In this example, in order to establish a suitable genome editing CRISPR system in diploid industrial Saccharomyces cerevisiae HD, the most reported CRISPR / Cas9 two-plasmid system in S. CEN / ARS element) to express Cas9 (pH-spCas9) (SEQ ID No. 1), and a multicopy plasmid (2μ plasmid) to express gRNA (p426H-LEU2-gRNA) (SEQ ID No. 2). Leucine deficiency occurs due to inactivation of the LEU2 gene on the S. cerevisiae genome, and only simultaneous inactivation of all alleles in polyploid yeast causes a leucine-deficient phenotype.

[0030] Using plasmid pRS426-SAM2 (SEQ ID No. 3) as a template, primers Leu-SAM2-For (SEQ ID No. 4) and Leu-SAM2-Rev (SEQ ID No. 5) were used to amplify 40 bp of LEU2 gene at both ends. The SAM2 gene expression cassette fragment of the source sequence was used as a homology repair template to perform LEU2 gene knock-in to examine the knock-in efficiency of t...

Example Embodiment

[0034] Example 2 Optimization of the concentration of gRNA plasmid and repair template DNA added

[0035] During the transformation process, different concentrations of gRNA and repair template have a greater impact on the efficiency of gene editing. Therefore, in this example, different concentrations of gRNA and repair template were used in the process of pRS42H-iCas9 and pKan100-LEU2-gRNA-mediated LEU2 gene knocking into the SAM2 expression cassette. The results are shown in Table 2. When the concentration of pKan100-LEU2-gRNA plasmid reaches 2.5 μg and the concentration of repair template containing SAM2 expression cassette reaches 5 μg, the gene knock-in efficiency can reach nearly 100%. Therefore, in subsequent gene editing, both gRNA and repair template are added with reference to this concentration.

[0036] Table 2 Comparison of different gRNA plasmid and repair template concentrations on the efficiency of LEU2 gene knock-in SAM2 expression cassette

[0037]

Example Embodiment

[0038] Example 3 Gene Knockout and Chromosome Fragment Deletion Based on CRISPR / Cas9 System

[0039] In this example, the optimized conditions were used to investigate the efficiency of gene knockout and chromosome deletion mediated by pRS42H-iCas9 and pKan100-GAL10-gRNA systems. Taking the GAL1, GAL7 and GAL10 regions on the chromosome of Saccharomyces cerevisiae as the target, using primers 1000bp-deletion-L-For (SEQ ID No. Template, amplify the 1000bp upstream homology arm fragment of the knockout genome, using primers 1000bp-deletion-R-For (SEQ ID No. 10) and 1000bp-deletion-R-Rev (SEQ ID No. 11), to Saccharomyces cerevisiae The genome is used as the template, and the 1000bp downstream homology arm fragment of the knockout genome is amplified, and the repair template DNA of the genome knockout 1000bp is obtained by fusion PCR; the same method is used, using the primer 2000bp-deletion-L-For (SEQ ID No.12) , 2000bp-deletion-L-Rev (SEQ ID No. 13), 2000bp-deletion-R-For (SEQ ...

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Abstract

The invention discloses a method for synthesizing S-adenosylmethionine from DL-methionine by modifying saccharomyces cerevisiae based on a CRISPR technology and an application. Genome modification methods of gene knock-in, gene knock-out and Delta site integration based on a CRISPR/Cas9 technology are constructed in diploid industrial saccharomyces cerevisiae; and furthermore, by utilizing the methods, DAAO and L-PheDH expression cassettes are knocked into an HPA3 gene locus of the saccharomyces strain, DAAO and L-PheDH expression cassettes are knocked into an SAH1 gene locus, and an SAM2 geneexpression cassette is knocked into an SPE2 gene locus, so that the capability of synthesizing the S-adenosylmethionine from the DL-methionine by obtained engineering bacteria is remarkably improved.

Description

technical field [0001] A method for transforming Saccharomyces cerevisiae using DL-methionine to synthesize S-adenosylmethionine based on CRISPR technology belongs to the field of bioengineering, and specifically relates to the construction of Saccharomyces cerevisiae CRISPR technology and a method for using DL-methionine to synthesize S-adenosylmethionine. [0002] technical background [0003] Saccharomyces cerevisiae is an important eukaryotic model organism widely used in the production of fuels, chemicals and recombinant proteins. Saccharomyces cerevisiae has excellent homologous recombination ability. Combined with the latest CRISPR system, it can perform genome editing efficiently and quickly. However, current research is mainly focused on the development of CRISPR tools for haploid laboratory yeast strains, such as BY4741 and BY4742. Less research has been done on tool development for polyploid industrial yeast strains with better tolerance, growth rate, and fermentat...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/113C12N15/90C12P19/40C12R1/865
CPCC12N15/113C12N15/902C12P19/40C12N2310/20
Inventor 徐志南刘伟连佳长黄磊
Owner ZHEJIANG UNIV
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