Reduced genome E. coli
Inventors
Campbell, John W. • Blattner, Frederick R. • Plunkett, Guy • Posfai, Gyorgy
Assignees
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Publication Number
US-9085765-B2
Publication Date
2015-07-21
Expiration Date
Abstract
Reduced genome strains of E. coli MGl 655 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 reduced-genome Escherichia coli strains having a genome with the nucleic acid sequence set forth in SEQ ID NO: 1 and comprising deletions of many listed genes. The disclosed gene deletions remove multiple genomic regions, including instability-related elements and proteins, so that the strain genome is defined both by SEQ ID NO: 1 and by the specific deletion set.
The document characterizes the reduced-genome E. coli strains for performance compared to parental or commercial strains, including growth rate, transformation efficiency, recombinant protein expression, and DNA production. The reported improvements also extend to genomic stability, including reduced IS hopping/mutation activity and improved DNA yield and quality.
The document further describes examples of using the reduced-genome strains for cloning and recombinant production, including cloning performance for difficult or toxic sequences and the presence or absence of an IS insertion in a specific example plasmid context. Exemplary assays include measuring growth, transformation efficiency, CAT expression as a model polypeptide, DNA yield, and genomic stability or mutation/IS activity, with detection of IS elements.
Claims Coverage
The independent claim defines a reduced-genome Escherichia coli strain with a specified genome sequence and a defined set of gene deletions. The dependent claims cover methods for expressing, introducing, and amplifying heterologous nucleic acids in the defined strain, including use of a vector.
Reduced-genome Escherichia coli strain defined by SEQ ID NO: 1 and extensive deletions
A strain of Escherichia coli having a genome with the nucleic acid sequence set forth in SEQ ID NO: 1 and comprising deletions of the following genes: b0245-b0301, b0303-b0310, b1336-b1411, b4426-b4427, b2441-b2450, b2622-b2654, b2657-b2660, b4462, b1994-b2008, b4435, b3322-b3338, b2349-b2363, b1539-b1579, b4269-b4320, b2968-b2972, b2975-b2977, b2979-b2987, b4466-4468, b1137-b1172, b0537-b0565, b0016-b0022, b4412-b4413, b0577-b0582, b4415, b2389-b2390, b2392-b2395, b0358-b0368, b0370-b0380, b2856-b2863, b3042-b3048, b0656, b1325-b1333, b2030-b2062, b2190-b2192, b3215-b3219, b3504-b3505, b1070-b1083, b1878-b1894, b1917-b1950, b4324-b4342, b4345-b4358, b4486, b0497-b0502, b0700-b0706, b1456-b1462, b3481-b3484, b3592-b3596, b0981-b0988, b1021-b1029, b2080-b2096, b4438, b3440-b3445, b4451, b3556-b3558, b4455, b1786, b0150-b0153, b2945, b2699, b0315-b0321, b0323-b0331, b0333-b0354, b2481-b2492, b2219-b2227, b4500, b2228-b2230, b3707-b3723, b0644-b0650, b4079-b4090, b4487, b4092-b4106, b0730-b0732, b3572-b3587, b1653, b2735-b2740, b2405-b2407, b3896-b3900, b1202, b4263-b4268, b0611, b2364-b2366, b0839, b0488-b0500, and b0502.
Polypeptide production using the defined reduced-genome strain with an operably linked construct
A method produces a specified polypeptide by providing a microorganism strain of claim 1, introducing a heterologous nucleic acid encoding the operably linked polypeptide, and culturing the transformed strain under conditions that express the polypeptide.
Introduction of a heterologous nucleic acid into the defined reduced-genome strain
A method introduces a heterologous nucleic acid into a microorganism by providing the microorganism strain of claim 1 and transforming the strain with the heterologous nucleic acid.
Delivery of heterologous nucleic acid as a vector for expression in the defined reduced-genome strain
The method wherein the heterologous nucleic acid is provided as a vector.
Amplification of a heterologous nucleic acid using the defined reduced-genome strain with a vector
A method amplifies a heterologous nucleic acid by providing a microorganism strain of claim 1, transforming it with a vector comprising the heterologous nucleic acid, and culturing the transformed strain under conditions suitable for amplification.
Overall, the claim set centers on a defined reduced-genome Escherichia coli strain specified by SEQ ID NO: 1 and extensive deletions, and extends to methods using that strain to introduce heterologous nucleic acids, deliver them via a vector, express an operably linked polypeptide, and amplify the heterologous nucleic acid.
Stated Advantages
Equal or improved performance versus parental or commercial strains in growth rate.
Equal or improved performance versus parental or commercial strains in transformation efficiency.
Equal or improved performance versus parental or commercial strains in recombinant protein expression.
Equal or improved performance versus parental or commercial strains in DNA production, including yield and quality.
Improved genomic stability, including reduced IS hopping/mutation activity.
Documented Applications
Production of expressed polypeptides using a reduced-genome Escherichia coli strain, with chloramphenicol acetyl transferase (CAT) used as a model polypeptide.
DNA production and evaluation of DNA yield and quality using the reduced-genome strains.
Cloning performance for difficult or toxic sequences, including an example involving IS insertion and absence.
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