阅读说明：本技术 一种获得串联酶功能缺失突变体的方法及应用 (Method for obtaining tandem enzyme function deletion mutant and application ) 是由 吴业春 祁幼林 靳海霞 于 2020-07-10 设计创作，主要内容包括：本发明涉及基因工程技术领域,公开了一种获得串联酶功能缺失突变体的方法,EPSPS功能缺失的酵母突变体的方法及应用。所述重组酵母菌株通过生物基因工程的方法改变了酵母的Aro1基因,获得了只有EPSPS功能缺失的酵母突变体。(The invention relates to the technical field of genetic engineering, and discloses a method for obtaining a tandem enzyme function deletion mutant, a method for preparing an EPSPS function deletion yeast mutant and application of the EPSPS function deletion yeast mutant. The recombinant yeast strain changes the Aro1 gene of the yeast by a biological genetic engineering method, and obtains a yeast mutant only with EPSPS function deletion.)

1. The method for obtaining the biological mutant with the function of the specific gene loss comprises the following steps:

1) on the sequence of the tandem polygene, the position of the gene segment related to the function of the specific gene is firstly determined,

2) deleting the sequence fragment or adding a base sequence with length of 3 times to the sequence fragment in organism by biotechnology, or changing only one or several bases in the sequence fragment,

3) the biological mutants are screened for the specific gene segments identified in the first step.

2. The method for obtaining the yeast mutant with the function deletion of the specific gene comprises the following steps:

1) on the sequence of the tandem polygene, the position of the gene segment related to the function of the specific gene is firstly determined,

2) biotechnologically deleting or adding a nucleotide sequence to the sequence fragment of the yeast, the length of the nucleotide sequence having to be a multiple of 3, or changing only one or several nucleotides of the sequence fragment,

3) the yeast mutants are screened for the specific gene segments identified in the first step.

3. The method for obtaining the EPSPS function-deleted yeast mutant comprises the following steps:

1) on the sequence of the tandem polygene, the position of the gene segment related to the EPSPS gene function is firstly determined,

2) biotechnologically deleting or adding a nucleotide sequence to the sequence fragment of the yeast, the length of the nucleotide sequence having to be a multiple of 3, or changing only one or several nucleotides of the sequence fragment,

3) the yeast mutants were screened on the basis of the EPSPS gene fragment determined in the first step.

4. The biotechnological method according to claim 1, 2 or 3 is gene editing.

5. The yeast mutants obtained in claim 1, 2 or 3 can be used for the functional identification of EPSPS in yeast, the comparison of EPSPS in yeast for glyphosate resistance identification, the directed evolution of EPSPS and its application in chemical and medical fields.

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to yeast mutants with corresponding functions produced by recombining and transforming yeast, and application of the yeast mutants in the fields of research and development, medicines, chemical industry and the like.

Background

Tandem enzymes (tandems enzymes) are proteins consisting of one polypeptide chain with two or more different catalytic activities, also called multifunctional enzymes (multifunctionality enzymes). It usually catalyzes a succession of reaction steps in the same metabolic pathway.

In order to study the function of a certain gene or to obtain a specific mutant form in an organism, a common practice is to create a loss-of-function mutant of the corresponding gene. For example, one or more base sequences may be inserted, deleted or altered in a DNA sequence encoding an enzyme or protein such that the DNA sequence of the gene does not properly encode a functional protein or produce an nonfunctional protein. If a deletion mutant is created in the above-described manner for a gene encoding a tandem enzyme, it is often difficult to simultaneously affect the activities of two or more enzymes connected in series, and it is difficult to obtain a mutant in which one of the enzymes is lost and the activities of the other enzymes are normal, which makes it difficult to study the function of one of the enzymes.

For example, the Aro1 protein encoded by Aro1 gene in yeast is a tandem enzyme with 5 enzyme functions, which has the catalytic activities of five enzymes in the pathway of synthesizing aromatic amino acids by shikimic acid pathway, and is respectively:

1. dehydroquinate synthase (3-dehydroquinate synthase, abbreviated DHQS, international classification No. EC4.2.3.4);

2. dehydroquinate dehydratase (3-dihydroquinate dehydratase, abbreviated DHQD, international class number EC 4.2.1.10);

3. shikimate dehydrogenase (Shikimate dehydrogenase, abbreviated SDH, international classification number EC1.1.1.25);

4. shikimate kinase (Shikimate kinase, abbreviated SK, international systematic classification number EC 2.7.1.71);

5.5-Enolpyruvylshikimate-3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase, or 5-Enolpyruvylshikimate-3-phosphate synthase, or EPSPsynthsase, abbreviated EPSPS, International Classification No. EC 2.5.1.19).

In yeast, due to the structural characteristics of the Aro1 gene, mutation or deletion of the Aro1 gene simultaneously affects the functions of the above-mentioned multiple other enzymes according to a conventional method, and thus a yeast mutant lacking only the EPSPS function cannot be obtained. Yeast is a unicellular eukaryote, and is often used as a model eukaryote for research and also for research and application fields of medicine, chemical engineering and the like due to convenient culture operation.

The invention aims to provide a method for obtaining a tandem enzyme mutant, a method for obtaining a mutant with only one enzyme function deletion, the mutant and application thereof in the fields of research and development, medicine, chemical industry and the like.

Disclosure of Invention

During the research and development process, the researchers of the invention also find that the method can be used for an Open Reading Frame (ORF) formed by connecting multiple genes with similar structures in series, and the biological mutant with the function of a specific gene being deleted can be obtained by utilizing the method of the invention without influencing the functions of other genes connected in series.

The invention aims to provide a method for obtaining a biological mutant with a specific gene function deletion, which is characterized in that the position of a gene fragment related to the specific gene function is determined on the sequence of a series of multiple genes, the sequence fragment is deleted or added with a base sequence by a biotechnology method, the length of the base sequence is required to be multiple of 3, or only one or a plurality of bases on the sequence fragment are changed to form a mutant gene, the mutant gene cannot correctly code the gene fragment, but cannot influence the coding of other functional gene sequences, and finally, a correct ORF can still be formed, and the functions of other genes can be completely preserved except the function deletion related to the specific gene fragment. So that the biological mutant with the function of the specific gene being deleted can be obtained.

The invention aims to provide a method for obtaining a yeast mutant with a specific gene function deletion, which comprises the steps of firstly determining the position of a gene segment related to the specific gene function on a series multigene sequence, deleting or adding a base sequence on the sequence segment by a biotechnology method, wherein the length of the base sequence is required to be multiple of 3, or only changing one or a plurality of bases on the sequence segment to form a mutant gene, wherein the mutant gene cannot correctly code the gene segment, but cannot influence the coding of other functional gene sequences, and finally, correct ORF can still be formed, and the functions of other genes except the function deletion related to the specific gene segment can be completely preserved. Thereby obtaining the yeast mutant with the function of the specific gene being deleted.

The invention aims to provide a method for obtaining EPSPS deleted yeast mutant, which comprises the steps of firstly determining the position of a gene segment related to the EPSPS function on the Aro1 gene, wherein the gene segment is the gene segment from 1198 to 2658 in the sequence of the gene Aro1, deleting or adding a base sequence of the sequence segment by a biotechnology method, wherein the length of the added or reduced base sequence is required to be multiple of 3, or only changing one or a plurality of bases on the sequence segment to form a mutant gene, wherein the mutant gene cannot correctly code the gene segment related to the EPSPS function, but does not influence the coding of other gene sequences, and finally, a correct ORF can still be formed, and the functions of other genes except the function deletion related to the EPSPS function can be completely preserved. Thus, a yeast mutant with EPSPS function deletion can be obtained.

In one embodiment of the present invention, EPSPS function deletion mutants are obtained by deleting 1461 nucleotides from positions 1198 to 2658 of the Aro1 gene (the nucleotide sequence of which is shown in SEQ ID NO: 1) in yeast by homologous recombination. The sequence of the finally obtained mutant Aro1 gene was changed as shown in SEQ ID No. 2.

In one embodiment of the present invention, EPSPS function deletion mutants are obtained by deleting all 1368 nucleotides from 1228 to 2595 of the Aro1 gene (the nucleotide sequence of which is shown in SEQ ID NO: 1) in yeast by homologous recombination. The sequence of the finally obtained mutant Aro1 gene was changed as shown in SEQ ID No. 3.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1: pCAS vector map

FIG. 2: YR11 vector diagram

FIG. 3: vector diagram of YCplac22 vector

FIG. 4: YR45 vector diagram

FIG. 5: EXAMPLE 1 comparison of the growth of the mutants in SC-H Medium

FIG. 6 comparison of the growth of the mutant of example 1 in SC-HFY Medium

FIG. 7: EXAMPLE 2 comparison of the growth of the mutants in SC-H Medium

FIG. 8: EXAMPLE 2 comparison of the growth of the mutants in SC-HFY Medium

Detailed Description

The present invention will be described in detail with reference to the following embodiments, but the scope of the invention is not limited thereto. All the modifications of the equivalent structure and the equivalent flow path made by the contents of the description and the drawings of the present invention, or the direct or indirect application to other related technical fields, are included in the scope of the present invention.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The present invention uses the most commonly used yeast strain BY4741 BY researchers as an example, and it is within the scope of the present invention to use other types of yeast strains.

YPAD medium: 10g/L of yeast extract, 20g/L of peptone, 0.1g/L of adenine sulfate and 20g/L of D-glucose. 20g/L agar powder is also added into the solid culture medium.

SC-H medium: YNB (ammonium sulfate-containing nitrogen source without amino yeast) 6.7g/L, D-glucose 20g/L, L-aspartic acid 0.05g/L, L-isoleucine 0.05g/L, L-methionine 0.05g/L, L-phenylalanine 0.05g/L, L-proline 0.05g/L, L-serine 0.05g/L, L-tyrosine 0.05g/L, L-valine 0.05g/L, L-adenine 0.1g/L, L-arginine 0.1g/L, L-cysteine 0.1g/L, L-histidine 0.1g/L, L-leucine 0.1g/L, L-lysine 0.1g/L, L-threonine 0.1g/L, L-tryptophan 0.1g/L, l-uracil 0.1g/L, pH 5.6. 20g/L agar powder is also added into the solid culture medium.

SC-HFY medium: 6.7g/L of ammonium sulfate-containing non-amino yeast nitrogen source (YNB), 20g/L of D-glucose, 0.05g/L of L-aspartic acid, 0.05g/L of L-isoleucine, 0.05g/L of L-methionine, 0.05g/L of L-proline, 0.05g/L of L-serine, 0.05g/L of L-valine, 0.1g/L of L-adenine, 0.1g/L of L-arginine, 0.1g/L of L-cysteine, 0.1g/L of L-leucine, 0.1g/L of L-lysine, 0.1g/L of L-threonine, 0.1g/L of L-tryptophan, 0.1g/L of L-uracil and pH 5.6. 20g/L agar powder is also added into the solid culture medium. (SC medium without three amino acids of L-histidine, L-phenylalanine and L-tyrosine).