Compositions and methods for amino acid biosynthesis
Inventors
Blattner, Frederick R. • Kim, Sun Chang • Lee, Jun Hyoung
Assignees
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Publication Number
US-8367380-B2
Publication Date
2013-02-05
Expiration Date
Abstract
The present invention relates generally to compositions and methods useful for, inter alia, production of commercial biologic products such as amino acids. More specifically, the present invention relates to genetically modified strains of microorganisms and the use thereof for the production of commercial products. The present invention also provides, inter alia, novel isolated DNA, nucleic acid, vectors and reduced genome bacteria.
Core Innovation
The invention provides recombinant/engineered reduced-genome bacteria for amino-acid biosynthesis centered on an E. coli relA mutant. The recombinant bacterium includes a genome containing a relA gene with at least one point mutation at position 547 or 548 of the relA coding sequence, where the mutation is selected from the nucleotide substitutions set forth in SEQ ID NO: 1 to SEQ ID NO: 4.
The document describes isolated DNA/nucleic acids and expression vectors containing SEQ ID NO: 1 to SEQ ID NO: 4, and embodiments of reduced genome bacteria carrying relA point mutations at positions 547 or 548. The reduced genome bacterium is defined with genome-size reduction relative to a native parent strain, including at least about 5% to about 10% smaller genome embodiments and other ranges described in the disclosure.
A biosynthesis method is described conceptually as biosynthesizing an amino acid using the reduced-genome bacterium by replacing a native relA with a mutant relA and expressing an amino acid. The disclosure further describes selecting the amino acid from a defined set of amino acids and includes an example involving threonine production in an engineered strain derived from MDS42, where a relA point mutation enhances threonine output.
Claims Coverage
The partial content identifies one independent claim directed to a recombinant bacterium, with dependent claims adding further quantitative and method limitations. The inventive coverage therefore focuses on the relA point mutations at defined positions, constrained nucleotide substitutions via SEQ ID NO: 1 to SEQ ID NO: 4, and reduced-genome bacterium embodiments, and then adds genome-size reduction and amino-acid biosynthesis method aspects through dependent claims.
Recombinant bacterium with relA point mutations at positions 547 or 548 defined by SEQ ID NO: 1-4
A recombinant bacterium comprising a genome containing a relA gene with at least one point mutation at position 547 or 548 of the relA coding sequence, where the mutation is selected from: the G-to-A mutation at position 547 in SEQ ID NO: 1, the G-to-T mutation at position 547 in SEQ ID NO: 2, the C-to-G mutation at position 548 in SEQ ID NO: 3, or the C-to-T mutation at position 548 in SEQ ID NO: 4.
Engineered genome-size reduction constraint versus native parent strain
A genetically engineered bacterium in which the genome is at least 5% smaller than the genome of its native parent strain.
Biosynthesizing an amino acid by expressing from the relA-mutant reduced-genome bacterium
A method of biosynthesizing an amino acid by providing the recombinant bacterium containing the relA point mutation and expressing an amino acid from the bacterium.
Choosing the amino-acid product from a defined amino-acid set
The method includes biosynthesizing an amino acid chosen from the specified set of amino acids including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Val, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, and Trp.
Plasmid-mediated delivery of biosynthetic genes under promoter control during amino-acid biosynthesis
A method in which an amino acid is biosynthesized by providing the specified bacterium, introducing a plasmid carrying biosynthetic genes, and expressing the amino acid from the bacterium under promoter control.
E. coli host specification
The recombinant bacterium is an E. coli bacterium.
Across the identified independent and dependent claim set in the partial content, the core claim coverage requires a recombinant bacterium with a relA gene carrying a point mutation at position 547 or 548 restricted to the specific nucleotide substitutions defined by SEQ ID NO: 1 to SEQ ID NO: 4. Dependent claims further require a quantitative reduced-genome constraint, and they frame amino-acid biosynthesis by expressing an amino acid from the engineered bacterium, with the product restricted to a defined amino-acid set and with the host specified as E. coli.
Stated Advantages
Documented Applications
Threonine production in engineered strains derived from MDS42 using a relA point mutation to enhance threonine output in engineered strains.
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