Riboswitch Screening for High L-Tryptophan Producing Microorganisms
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Solution Overview
Problem
Current methods for screening high L-tryptophan-producing microorganisms are inefficient and limited in throughput, particularly for large strain libraries, and lack applicability to various target metabolites.
Innovation Solution
A riboswitch system incorporating a tryptophan aptamer and a selectable marker gene, such as tetA, is used to selectively screen for high L-tryptophan-producing microorganisms by regulating gene expression based on L-tryptophan concentration, allowing for quick and efficient identification of strains with high L-tryptophan production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If liquid/gas chromatography is used to analyze metabolite concentrations in individual strains, then measurement precision is improved, but productivity deteriorates due to low throughput capability
Solution Approach 1:
The patent divides the strain library into multiple sub-libraries and processes them in parallel using multiple multiplate readers simultaneously. Each multiplate reader analyzes a subset of strains, enabling concurrent processing of large numbers of strains while maintaining precise metabolite concentration measurements through quantitative analysis of chromogenic reactions.
Solution Approach 2:
The patent uses a chromogenic substrate that produces a detectable signal copy of the metabolite concentration information. The substrate is converted into a detectable product (e.g., colored compound) that can be measured optically, creating an amplifiable signal copy that enables sensitive detection of metabolite concentrations in many strains simultaneously.
2Productivity
If multiplate methods are used to analyze metabolites in partitioned wells, then productivity is improved through simultaneous analysis of multiple strains, but measurement precision deteriorates due to limited detection sensitivity
Solution Approach 1:
The patent changes the detection parameter from direct metabolite detection to detection of a chromogenic product. By using substrates that produce colored compounds upon metabolite conversion, the system achieves enhanced optical signal intensity that can be detected with standard multiplate readers, thereby improving detection sensitivity while maintaining high throughput.
Solution Approach 2:
The patent introduces a chromogenic substrate as an intermediary between the metabolite and the detection system. The substrate serves as a mediator that converts the metabolite into a detectable signal, amplifying the measurement capability and enabling sensitive detection of metabolite concentrations in high-throughput screening.
3Productivity
If fluorescence-activated cell sorting is used to detect fluorescent metabolites, then productivity is improved through rapid detection of large cell populations, but adaptability deteriorates since it applies only to fluorescent metabolites
Solution Approach 1:
The patent develops a chromogenic detection system that can be applied to various metabolite types through the use of different chromogenic substrates. The same multiplate reader infrastructure can detect different metabolites by simply changing the substrate, providing universal applicability across multiple metabolite classes while maintaining high-throughput capability.
Solution Approach 2:
The patent changes the detection mechanism from fluorescence-based to chromogenic-based detection. This parameter change allows the system to detect a broader range of metabolites that do not inherently fluoresce, by using substrates that produce colored compounds upon metabolite conversion, thereby enhancing adaptability while preserving high productivity.
4Adaptability or versatility
If conventional mutagenesis methods are used to produce strain libraries, then adaptability is improved through diverse mutation types, but device complexity increases due to multiple mutagenesis techniques required
Solution Approach 1:
The patent combines multiple mutagenesis techniques (UV irradiation, chemical mutagenesis, genome shuffling, transposon insertion) into a single integrated strain library production protocol. By merging these diverse methods to create a comprehensive strain library, the system achieves high adaptability and diversity while managing complexity through unified processing and screening approaches.
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
This approach enables rapid and effective selection of strains producing high concentrations of L-tryptophan, enhancing the price competitiveness of tryptophan production by improving screening efficiency and applicability across various metabolites.
Implementation Method 1
a riboswitch for screening a high L-tryptophan-producing microorganism, which includes a tryptophan aptamer
Implementation Method 2
a DNA sequence consisting of 1 to 20 nucleotides and a selectable marker gene
Data Source
AI summary
A method of screening a high L-tryptophan-producing microorganism using a riboswitch is provided. More particularly, a riboswitch for screening a high L-tryptophan-producing microorganism including a tryptophan aptamer, a DNA sequence consisting of 1 to 20 nucleotides and a selectable marker gene, and a method of screening a high L-tryptophan-producing microorganism using the same are provided. The riboswitch and the method of screening a high L-tryptophan-producing microorganism using the same can be useful in selecting a strain producing a high concentration of L-tryptophan in a relatively quick and easy manner, and thus enhancing price competitiveness of tryptophan production using microorganisms.