Method for regulating gene expression intensity on microbial chromosome by using artificial regulatory element and its library

Abstract

The invention discloses a method for regulating the expression intensity of genes on microorganism chromosomes by using artificial regulatory elements and libraries thereof. The method for regulating the gene expression intensity on the microbial chromosome by using the artificial regulatory element and its library provided by the present invention includes the following steps: amplify a DNA fragment by PCR, which includes the upstream sequence of the original regulatory region of the gene to be regulated on the microbial chromosome A homologous fragment containing 40-3000 bases, resistance marker gene, artificial regulatory element library or an artificial regulatory element with specific expression strength, homologous to the sequence downstream of the start codon of the gene to be regulated on the microbial chromosome A fragment containing 40-3000 bases; the DNA fragment is electrotransformed into the microorganism, and the original regulatory region of the gene to be regulated on the microbial chromosome is replaced by an artificial regulatory element library or an artificial regulatory element with a specific expression intensity .

Description

technical field [0001] The invention relates to a method for regulating the expression intensity of genes on microorganism chromosomes by using artificial regulatory elements and libraries thereof. Background technique [0002] Gene expression regulation technology is one of the core technologies of metabolic engineering. At present, when transforming microorganisms to improve the synthesis ability of target products, a commonly used strategy is to regulate the expression of genes through the method of plasmid overexpression, and increase the activity of one or several key enzymes, thereby enhancing the metabolism of microorganisms to synthesize target products flow. When the plasmid is overexpressed, inducible promoters (such as lac, trc, ara and other promoters) are usually used to regulate the expression of the gene. However, this approach has three major drawbacks. First, the use of these inducible promoters in the production of bulk chemicals is not economically feas...

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Problems solved by technology

However, this approach has three major drawbacks

First, the use of these inducible promoters in the production of bulk chemicals is not economically feasible due to the need to add expensive inducers such as lactose, IPTG, arabinose, etc. to induce the expression of these promoters

Second, plasmid-based expression has many disadvantages: the maintenance of plasmids will cause a large metabolic load on host cells, especially high-copy plasmids; the genetic stability of many plasmids is not good; only the replication of low-copy number plasmids and cell The reproduction of the cells is synchronized, so only they maintain the same copy number in all cells; the synthesis of many compounds requires the construction of a complex metabolic pathway in the cell, so it is necessary to introduce long DNA fragments containing multiple genes, Most plasmids are more difficult to carry long DNA fragments

At present, there are relatively few reports on the construction of promoter upstream region, messenger RNA stability region and ribosome binding site regulatory elements

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