Synthetic sRNA for Dynamic Gene Expression Control
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Solution Overview
Problem
Conventional gene deletion methods in metabolic engineering are time-consuming, cause complete loss of gene function, and are difficult to restore, limiting the ability to efficiently regulate gene expression and optimize metabolic flux for producing desired metabolites while maintaining cell growth.
Innovation Solution
Development of a novel, short-length customized synthetic sRNA comprising an Hfq binding site derived from MicC and a region that base-pairs with target gene mRNA, allowing for efficient regulation of gene expression without modifying the chromosomal sequence, and can be easily applied to various strains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional gene deletion method is used, then gene function is completely lost, but gene expression cannot be regulated to desired level and cell growth is inhibited
Solution Approach 1:
The patent applies dynamics by making the gene silencing effect adjustable rather than fixed. The sRNA expression level can be controlled to achieve desired silencing intensity, allowing dynamic regulation between complete gene loss and partial expression, thereby maintaining cell growth while achieving metabolic flux optimization.
Solution Approach 2:
The patent changes the parameter of gene expression from binary (on/off) to continuous (variable levels). By controlling sRNA expression levels and designing sRNAs with different binding affinities, the invention enables precise regulation of target gene expression to achieve optimal balance between metabolic flux redirection and cell growth maintenance.
2Productivity
If conventional gene deletion method is used, then metabolic flux is blocked, but time required for gene deletion is long (one week or longer)
Solution Approach 1:
The patent applies preliminary action by pre-designing sRNA sequences with optimized binding sites and structures. The sRNA can be synthesized and introduced directly into cells, eliminating the need for time-consuming chromosomal manipulation steps such as homologous recombination and antibiotic resistance gene removal, thus achieving rapid metabolic flux regulation.
Solution Approach 2:
The patent replaces the mechanical chromosomal DNA manipulation system with an RNA-based regulatory system. Instead of physically modifying the chromosome through recombination enzymes, the invention uses introduced sRNA molecules that bind to target mRNA to achieve gene silencing, dramatically reducing the time required for metabolic engineering.
3Ease of manufacture
If conventional gene deletion method is used, then gene is deleted from chromosome, but gene is difficult to restore and cannot be applied to other strains
Solution Approach 1:
The patent applies copying by using synthetic sRNA sequences that can be replicated and transferred across different strains. The sRNA design includes conserved structural elements and binding sites that can be copied from one strain to another, enabling the same gene silencing strategy to be applied universally across different prokaryotic strains without repeating the entire deletion process.
Solution Approach 2:
The patent achieves universality by designing sRNA molecules with modular structures where the target-specific binding region can be easily changed while maintaining the core functional elements. This allows the same sRNA platform to be applied to different target genes and different strains, providing a universal tool for metabolic engineering across prokaryotic systems.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The synthetic sRNA effectively inhibits target gene expression, mimicking gene deletion effects, while allowing for fine-tuned regulation of metabolic flux, enhancing production of desired metabolites like tyrosine and cadaverine, and maintaining cell growth.
Implementation Method 1
a region that base-pairs with the target gene mRNA
Implementation Method 2
an Hfq binding site from the sRNA of MicC
Data Source
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AI summary
The present invention relates to a novel customized sRNA that reduces gene expression in prokaryotic cells, a preparation method thereof, and the use thereof, and more particularly to a synthetic sRNA comprising an Hfq binding site, derived from the sRNA of any one of MicC, SgrS and MicF, and a region that base-pairs with the target gene mRNA, and to a preparation method thereof and the use thereof. The synthetic sRNA according to the invention has an advantage in that the degree of inhibition of the target gene can be controlled by regulating the ability of the synthetic sRNA to bind to the mRNA of the target gene. The use of the synthetic sRNA that regulates the expression of the target gene makes it possible to effectively construct a recombinant microorganism without using a conventional gene deletion method and to reduce the expression of the target gene, and thus the synthetic sRNA is useful for the production of recombinant microorganisms. Also, the synthetic sRNA can be quickly applied to various strains, and thus is very suitable for the measurement of metabolic capabilities of strains and the selection of the most suitable strain. In addition, recombinant microorganisms, which are obtained by metabolic flux manipulation using the synthetic sRNA and produce tyrosine or cadaverine with high efficiency, are useful in the drug and industrial fields. In other words, the use of the sRNA according to the present invention can make it easy to select target genes whose expression is to be inhibited for the highly efficient production of metabolites. Accordingly, the synthetic sRNA can be used to construct recombinant strains for efficient production of various metabolites and to establish efficient methods for production of various metabolites, and thus is highly useful.