Reduced genome E. coli
Inventors
Campbell, John W • Blattner, Frederick R. • Plunkett, Guy • Posfai, Gyorgy
Assignees
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Publication Number
US-8178339-B2
Publication Date
2012-05-15
Expiration Date
Abstract
Reduced genome strains of E. coli MG1655 are described. In various embodiments, the strains have one or more of equal or improved growth rate, transformation efficiency, protein expression, DNA production, DNA yield and/or DNA quality compared to the parental strain and commercially available strains.
Core Innovation
The invention relates to an engineered strain of E. coli having a genome with the nucleic acid sequence set forth in SEQ ID NO: 1. The genome is defined by having a set of genes deleted therefrom, with the deletions specified as gene regions removed from the genome, and the E. coli strain is provided as a reduced-genome strain derived from MG1655.
The reduced-genome E. coli strain is described as useful for improving industrial and research performance. The document reports equal or improved growth rate, higher transformation efficiency, comparable recombinant protein expression relative to MG1655, increased recombinant DNA yield, and reduced mutation frequency from IS hopping, described in terms of improved DNA stability and quality.
The document additionally describes improved cloning performance, including an improved ability to clone otherwise difficult sequences, including toxic sequences. The examples include reduced-genome strains such as MDS41–MDS60, including MDS42recA−, which are described as having specific genomic regions deleted that correspond to categories such as restriction-modification genes, insertion sequence (IS) elements, and other deletable gene/function regions.
Claims Coverage
The claims include one independent claim directed to a specific engineered E. coli deletion strain, with dependent claims refining use in cell-engineering workflows and delivering heterologous nucleic acids by transformation. The inventive subject matter centers on the defined reduced-genome genotype (SEQ ID NO: 1 plus specified deleted gene regions) and its use for polypeptide production, heterologous nucleic acid introduction, and heterologous nucleic acid amplification.
Defined reduced-genome E. coli deletion strain
A strain of E. coli having a genome with the nucleic acid sequence set forth in SEQ ID NO: 1 with specified genes deleted therefrom.
Polypeptide production using the deletion strain
A method for producing a polypeptide by providing the claim 1 strain, transforming it with a heterologous nucleic acid encoding the polypeptide operatively linked to an expression control sequence, and culturing under conditions suitable to express the polypeptide.
Introduction of a heterologous nucleic acid by transformation
A method of introducing a heterologous nucleic acid into a microorganism by providing the claim 1 strain and transforming the strain with the heterologous nucleic acid.
Vector-based heterologous nucleic acid delivery
The method wherein the heterologous nucleic acid is a vector.
Amplification of a heterologous nucleic acid using the deletion strain
A method of amplifying a heterologous nucleic acid by providing the claim 1 strain, transforming the strain with a vector comprising the heterologous nucleic acid, and culturing under conditions suitable to amplify the heterologous nucleic acid.
Overall, the claims are grounded in a defined reduced-genome E. coli strain characterized by SEQ ID NO: 1 with specified gene deletions, and extend to methods that use the strain for transformation-based introduction of heterologous nucleic acids, including polypeptide production and amplification, including vector-based delivery.
Stated Advantages
Equal or improved growth rate.
Higher transformation efficiency.
Comparable recombinant protein expression.
Increased recombinant DNA yield.
Reduced mutation frequency from IS hopping, described as improved DNA stability and quality.
Improved ability to clone otherwise difficult sequences, including toxic sequences.
Documented Applications
Producing a polypeptide using the deletion strain with a heterologous nucleic acid encoding the polypeptide operatively linked to an expression control sequence and culturing under conditions suitable to express the polypeptide.
Introducing a heterologous nucleic acid into a microorganism by providing the deletion strain and transforming with the heterologous nucleic acid.
Amplifying a heterologous nucleic acid by transforming the deletion strain with a vector comprising the heterologous nucleic acid and culturing under conditions suitable to amplify it.
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