Method of Identifying a Cell with an Intracellular Concentration of a Specific Metabolite, Which Intracellular Concentration Is Increased in Comparison with the Cell's Wildtype, Where the Modification of the Cell Is Achieved by Recombineering, and Method of Making a Production Cell with Optimized Production of a Specific Metabolite, Which Production Cell Is Genetically Modified in Comparison with Its Wildtype, Method of Producing This Metabolite, and Nucleic Acids Which Are Suitable Therefor

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This patent describes a method for rapidly engineering and identifying genetically modified cells (especially bacteria) that have an increased intracellular concentration of a desired metabolite, such as an amino acid or vitamin. The core invention combines recombineering (a technique for targeted genetic modification using recombinase enzymes) with metabolite sensors (which detect and signal the presence of the target metabolite through fluorescence). This allows for the high-throughput selection and isolation of cells that are optimized to produce higher amounts of the target compound, without the need for laborious, traditional plating and screening processes.

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• Industrial-scale microbial production of amino acids (e.g., L-lysine), vitamins, organic acids, fatty acids, or plant-derived compounds.
• Development of genetically optimized microbial strains for efficient synthesis of bio-based chemicals and pharmaceuticals.
• Screening and identification of new metabolic pathways or beneficial mutations in microorganisms.
• Improving yields in fermentation processes for food, feed, or biofuel industries.
• Research applications in metabolic engineering and synthetic biology for pathway optimization.

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• Enables rapid and targeted genetic improvement of production strains, significantly shortening time to market.
• Allows high-throughput screening and selection of high-producing cell variants using fluorescence-based sensors, increasing efficiency and accuracy.
• Reduces the need for traditional, labor-intensive methods like plating and colony picking, thus saving labor and resources.
• Can be adapted for a wide variety of metabolites and organisms beyond model bacteria, expanding its applicability.
• Facilitates the discovery and transfer of beneficial genetic mutations to industrial strains, leading to enhanced production yields and cost-effectiveness.

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Patent Abstract

The invention relates to a method of identifying a cell with an intracellular concentration of a specific metabolite, which intracellular concentration is increased in comparison with the cell's wildtype, where the modification of the cell is achieved by recombineering, and a method of making a production cell with optimized production of a specific metabolite, which production cell is genetically modified in comparison with its wildtype, a method of producing this metabolite, and nucleic acids which are suitable therefor. In accordance with the invention, a gene which codes for a recombinase and which is homologous to a known recombinase gene is transformed into a cell via a vector, and a DNA which comprises at least one modified gene G1 to Gn or at least one mutation M1 to Mn is introduced into this cell, and the cell with the highest metabolite production is identified by means of metabolite sensors. The mutation which is made responsible for the increased production is isolated from this cell, and the gene or the mutation is recovered and introduced into a production strain which thereby shows an increased production of the metabolite.