Pyruvate Carboxylase and DNA Encoding Pyruvate Carboxylase, Plasmid Containing the DNA, and Microorganism for Production and Method for Producing Products Whose Biosynthesis Includes Oxaloacetate as a Precursor, Chromosome and Screening Method

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This patent relates to genetically modified pyruvate carboxylase enzymes and their encoding DNA, specialized plasmids carrying these genes, and microorganisms that express them. The key innovation is a specific mutation (T132A: threonine at position 132 replaced with alanine) in pyruvate carboxylase, which, when expressed in microbial production strains, results in a significantly increased yield (7-19% higher) of products whose biosynthesis requires oxaloacetate as a precursor, such as important amino acids and metabolic intermediates. The patent also introduces a novel screening method for identifying such improved enzyme variants.

Use CasesContent extracted from patent full text and abstract with AI.

• Industrial-scale microbial production of amino acids such as L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine, and L-methionine.
• Biotechnological manufacture of other compounds derived from the citric acid cycle, e.g., succinate, malate, fumarate, citrate, isocitrate, and 2-oxoglutarate.
• Enhanced fermentation processes for producing diamines (e.g., putrescine, cadaverine) and related metabolic chemicals.
• Screening and development of high-yield production strains for the biotech and industrial fermentation sectors.
• Production of value-added chemicals like itaconate, butanol, 1-propanol, gamma-aminobutyric acid (GABA), ectoine, and others that use oxaloacetate-derived biosynthetic pathways.

BenefitsContent extracted from patent full text and abstract with AI.

• Higher yield of desired metabolic products (7–19% increase demonstrated) leads to improved process economics and industrial competitiveness.
• Applicable to a broad range of microorganisms (Corynebacterium, E. coli, yeast) used in biotech manufacturing.
• Enables more efficient use of feedstocks and resources in fermentation processes.
• Reduces product inhibition and bottlenecks by overcoming natural feedback inhibition in pyruvate carboxylase.
• Facilitates rapid screening and optimization of enzyme variants and production strains through a fluorescence-based high-throughput method.
• Contributes to sustainable bioproduction and supports the shift towards greener chemical and amino acid manufacturing.

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Patent Abstract

The invention relates to a pyruvate carboxylase and a pyruvate carboxylase-encoding DNA, a plasmid containing said DNA and a microorganism for the production thereof, and to methods for the production of products the biosynthesis of which includes oxaloacetate as a precursor, and to a chromosome and a screening method. The invention makes a DNA available that codes for a pyruvate carboxylase having an at least 70% sequence identity with sequence no. 1 and where the triplet that codes for threonine-132 is replaced by an alanine-encoding triplet in positions 394-396. The expression of said gene results in a pyruvate carboxylase by means of which the yield of the reaction of metabolic products derived from oxaloacetate is between 7 - 19 % higher with respect to the strain containing the native Pyc. The claimed screening method enables a claimed pyruvate carboxylase to be found.