Method for Reducing Misincorporation of Non-Canonical Branched-Chain Amino Acids

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This invention provides a method to produce recombinant proteins (such as therapeutic proteins) in microbial host cells—most notably engineered E. coli—by modulating specific enzymatic activities in the cell. The technique reduces the unintended inclusion (misincorporation) of non-canonical branched-chain amino acids (such as norleucine, norvaline, and beta-methylnorleucine) into the target protein, which can compromise purity and biological activity. By altering the activity of certain enzymes involved in amino acid biosynthesis, the method ensures higher fidelity and quality in the produced proteins.

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• Manufacture of therapeutic proteins, including insulin, proinsulin, antibodies, and other biopharmaceuticals, where high purity and correct amino acid composition are critical.
• Large-scale microbial fermentation processes for recombinant protein production in the pharmaceutical or biotechnology industries.
• Production of high-fidelity research proteins for structural and functional studies, where minimal amino acid misincorporation is required.
• Production of enzymes or industrial proteins with strict requirements for sequence fidelity.

BenefitsContent extracted from patent full text and abstract with AI.

• Significantly reduces unwanted incorporation of non-standard amino acids into recombinant proteins, improving product homogeneity and safety.
• Ensures higher biological activity and lower immunogenicity for therapeutic proteins.
• Decreases the need for extensive downstream purification steps aimed at removing misincorporated proteins, reducing costs and increasing yield.
• Enables consistent manufacturing of biopharmaceuticals that must comply with strict regulatory standards for protein sequence fidelity.
• Applicable to a variety of proteins and microbial hosts, offering broad utility across multiple biotechnology applications.

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Patent Abstract

The present invention relates to a method for producing a recombinant polypeptide of interest in a microbial host cell, comprising (a) introducing a polynucleotide encoding the polypeptide of interest into a microbial host cell which has been modified such that an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase activity (EC 4.3.1.19) is modulated in said microbial host cell as compared to the enzymatic activity in an unmodified microbial host cell, and (b) expressing said polypeptide of interest in said microbial host cell. Moreover, the present invention relates to a method for reducing misincorporation of at least one non-canonical branched-chain amino acid into a recombinant polypeptide of interest expressed in a microbial host cell.