Pyruvate-Responsive Biosensor for Metabolic Flux Control
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Solution Overview
Problem
Traditional metabolic engineering techniques face challenges such as imbalanced intracellular metabolic flux, low raw material utilization, and accumulation of toxic metabolites, making it difficult to maximize compound titer and productivity, and existing pyruvate biosensors lack the ability to precisely control metabolic pathways in microbial networks.
Innovation Solution
Construction of pyruvate-responsive biosensors in Bacillus subtilis using the PdhR gene, P43 promoter, and egfp reporter gene, with a PdhR binding sequence inserted at specific sites, allowing for precise control of gene expression and dynamic regulation of intracellular metabolic flux.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional metabolic engineering techniques (overexpression of genes, knockout of pathways) are used to increase compound production, then productivity is improved, but intracellular metabolic flux becomes imbalanced and toxic metabolites accumulate
Solution Approach 1:
The patent employs a pyruvate-responsive biosensor that utilizes feedback regulation to dynamically adjust gene expression levels based on intracellular pyruvate concentration. The biosensor contains a pyruvate-responsive promoter that activates transcription of target genes when pyruvate levels reach a threshold, creating a self-regulating system that prevents metabolic flux imbalance and toxic metabolite accumulation while maintaining high productivity
Solution Approach 2:
The invention transforms static metabolic engineering approaches into dynamic control systems. By introducing a pyruvate-responsive element upstream of the promoter, the system can dynamically respond to changing intracellular pyruvate levels and adjust gene expression accordingly, enabling real-time optimization of metabolic flux distribution
2Adaptability or versatility
If traditional mutagenesis breeding methods are used to generate mutant strains, then strain diversity is improved, but screening efficiency decreases due to high cost and low throughput of detection methods
Solution Approach 1:
The patent employs fluorescent reporter genes (gfp, mcherry) that produce visible color changes in response to pyruvate levels, enabling rapid visual screening of mutant strains. This allows high-throughput identification of high-producing strains by simply observing fluorescence intensity, dramatically increasing screening throughput compared to traditional HPLC-MS methods
Solution Approach 2:
The biosensor system creates a visible copy (fluorescence signal) of the intracellular pyruvate level, which correlates with product production. This allows indirect but rapid assessment of strain performance through fluorescence intensity rather than direct measurement of product concentration, enabling high-throughput screening
3Measurement precision
If existing pyruvate biosensors are used for metabolic monitoring, then detection capability is improved, but precise control of metabolic pathways is insufficient
Solution Approach 1:
The patent merges the detection function (pyruvate-responsive biosensor) with the control function (promoter-driven gene expression) into an integrated system. The pyruvate-responsive element upstream of the promoter directly links pyruvate level detection to gene expression control, enabling precise and straightforward metabolic pathway regulation without complex additional components
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 biosensors achieve a dynamic range of 0.6 to 30.7 times increase in expression levels, providing good specificity and response to pyruvate concentrations, enabling precise control of intracellular metabolic flux and avoiding premature accumulation of toxic compounds.
Implementation Method 1
transcriptional regulators are a class of proteins or RNAs in an organism that can respond to specific signals and regulate the expression of related genes in the cell
Implementation Method 2
PdhR is a naturally occurring pyruvate-responsive transcriptional regulator
Implementation Method 3
the reporter gene egfp
Data Source
AI summary
The present invention provides a pyruvate-responsive biosensor and a construction method and use thereof. In the present invention, pyruvate-responsive biosensors with different dynamic ranges are successfully constructed by optimizing the PdhR binding sequence inserted on the P43 promoter and optimizing the insertion site, wherein the minimum increase in dynamic range is by 0.6 time, and the maximum increase is by 30.7 times. The pyruvate-responsive biosensors are useful in the precise control of the expression of each gene in the cell. Since pyruvate is a key metabolite of central carbon in the cells, these biosensors are capable of dynamically regulating the expression level of intracellular genes according to changes in the content of pyruvate in the cells, thereby achieving the dynamic control of intracellular metabolic flux. The pyruvate biosensor obtained in the present invention has a good specificity, and a response range to pyruvate of 10-35 nmol/g DCW.