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Method for simultaneously transferring multiple genes into microbial genome

A technology of microorganisms and genomes, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of difficult transformation, difficult to recombine strains, low gene copy number, etc., and achieve the effect of avoiding trouble

Inactive Publication Date: 2012-10-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

However, when there are too many expressed genes, there are the following limitations: on the one hand, it is difficult to construct the vector, and multiple connection transformations are required; on the other hand, if the vector is too large, the transformation is difficult, and it is not easy to obtain recombinant strains; Vectors, gene copy numbers are often low, and it is difficult to achieve high-efficiency expression of foreign genes

Method used

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  • Method for simultaneously transferring multiple genes into microbial genome
  • Method for simultaneously transferring multiple genes into microbial genome
  • Method for simultaneously transferring multiple genes into microbial genome

Examples

Experimental program
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Embodiment 1

[0028] Example 1. Construction of xylose isomerization pathway in Saccharomyces cerevisiae

[0029] In this example, xylose isomerase gene (XI gene) and xylulokinase gene (XK gene) are expressed in S. The integration site is a multi-copy 18srDNA integration site.

[0030] 1. Obtaining the xylose isomerase gene expression cassette

[0031] Artificially synthesized xylose isomerase gene expression cassette (also known as C1 fragment, 3047bp), which is the double-stranded DNA molecule shown in sequence 1 of the sequence table (in sequence 1, the 1st to 766th nucleotides from the 5' end are Homologous sequence A of Saccharomyces cerevisiae 18srDNA, the 767th to 1444th nucleotides are the GPD promoter, the 1445th to 2758th nucleotides are the XI gene, and the 2759th to 3047th nucleotides are the CYC terminator). Wherein, the XI gene encodes the protein shown in sequence 4 of the sequence listing.

[0032] 2. Acquisition of Xylulokinase Gene Expression Cassette

[0033]Artificia...

Embodiment 2

[0046] Embodiment 2, expression NADH oxidase gene in Saccharomyces cerevisiae

[0047] In this embodiment, the NADH oxidase gene (noxE gene) is expressed in the recombinant bacteria obtained in Example 1 to reduce the production of xylitol in xylose fermentation; in order to increase the copy number, this case selects 26srDNA in Saccharomyces cerevisiae as gene integration location.

[0048] 1. Construction of NADH oxidase gene expression cassette

[0049] Artificially synthesized NADH oxidase gene expression cassette (also known as C4 fragment, 3460bp), which is the double-stranded DNA molecule shown in sequence 7 of the sequence table (the 1st to 1180th nucleotides from the 5' end of sequence 7 are Saccharomyces cerevisiae 26srDNA Homologous sequence A, the 1181st to 1778th nucleotide is the PGK promoter, the 1779th to 3119th nucleotide is the noxE gene, and the 3120th to 3460th nucleotide is the AOX terminator). Wherein, the noxE gene encodes the protein shown in sequence...

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Abstract

The invention discloses a method for simultaneously transferring multiple genes into microbial genome. The invention provides a method for preparing recombinant microorganism expressing multiple exogenous genes; the method comprises the following steps of: transferring expression cassettes of all genes into the host microorganism to obtain the recombinant microorganism combining multiple exogenous genes into the genome and expressing the multiple exogenous genes, wherein the 5' end of the expression cassette of the first gene has a homologous arm A, the 3' end of the expression cassette of the last gene has a homologous arm B, and the 3' tail end of the expression cassette of each gene and the 5' tail end of the expression cassette of the next gene have the same homologous arm; and the homologous arm A and the homologous arm B can be subjected to homologous recombination with the genome of the host microorganism. Through the method, multiple genes contained in a target metabolic pathway can be transferred into the host strain at one step and arranged according to a set sequence, the required engineering strain is directly obtained, and the troubles caused by multiple times of transformation and vector construction are avoided.

Description

technical field [0001] The invention relates to a method for simultaneously introducing multiple genes into the genome of microorganisms. Background technique [0002] Energy, population and environmental problems are the main problems at present. If the abundant renewable resources are converted into bio-energy, not only the problems of energy and environment will be solved, but also food will be saved and the food problem caused by the large population will be solved. [0003] At present, genetic engineering transformation has become the core technology to improve the level of industrial microbial strains and promote the industrialization of biotechnology. An important direction of genetic engineering transformation is to introduce new genes or increase the expression of original genes in host bacteria, which can be achieved by transforming host bacteria with expression vectors containing target gene segments, or by inserting target gene segments into recombinant cells. T...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N1/19C12R1/865
Inventor 张延平张博孙红兵朱泰承李寅马延和
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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