Reduced genome bacteria with improved genetic stability
Inventors
Blattner, Frederick R. • Csorgo, Balint • Posfai, Gyorgy
Assignees
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Publication Number
US-9340791-B2
Publication Date
2016-05-17
Expiration Date
Abstract
Reduced genome bacteria with improved genetic stability are provided. Also provided are methods of producing polypeptides using the reduced genome bacteria with improved genetic stability.
Core Innovation
The invention relates to reduced-genome Escherichia coli bacteria engineered for improved genetic stability. The bacteria have a genome that is genetically engineered to be up to 22% smaller than the genome of a native parent strain, including reduced-genome backgrounds such as E. coli strains including MDS42 and MG1655.
The bacteria are further defined by genetic engineering that removes multiple insertion sequences and eliminates error-prone DNA replication and repair functions. In particular, the bacteria lack specified insertion sequences (IS1, IS2, IS3, IS4, IS5, IS30, IS150, IS186, IS600, IS911 and IS10) and lack functional polB and dinB genes, with optional lack of functional umuDC (polV/umuDC) gene.
The reduced-genome, polymerase-deficient bacteria are described as having very low mutation rates and improved mutation-rate behavior under stress conditions. Documented examples include reduced spontaneous mutation rates in fluctuation assays using cycA mutation assays, reduced mutation-rate response under stresses including mitomycin C and protein overexpression, and delayed accumulation of loss-of-function mutations for a toxic gene carried on a plasmid using a SinI/SinI methyltransferase system.
Claims Coverage
The claims cover one main inventive concept with several inventive features, including reduced genome size, removal of specified insertion sequences, and deletion of error-prone polymerase genes polB and dinB, optionally umuDC. Dependent claims further narrow the native parent strain, specify a non-functional umuDC gene, define deleted DNA segments of MG1655, and include use of the engineered bacterium to express and collect an encoded polypeptide.
Up to 22% reduced-genome E. coli bacterium
A reduced genome Escherichia coli bacterium, having a genome that is genetically engineered to be up to 22% smaller than the genome of its native parent strain.
Loss of specified insertion sequences
A bacterium that lacks all IS1, IS2, IS3, IS4, IS5, IS30, IS150, IS186, IS600, IS911 and IS10 insertion sequences.
Non-functional polB and dinB and optionally umuDC
A bacterium that lacks a functional polB gene and lacks a functional dinB gene, wherein the bacterium optionally lacks a functional umuDC gene.
Deleted DNA segments of MG1655 K-12
A bacterium having deleted therefrom at least the following DNA segments of the E. coli K-12 strain MG1655: 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 and b2945.
Native parent strain is MG1655 K-12
Wherein said native parent strain is K12 strain MG1655.
UmuDC is non-functional
Wherein said bacterium has a non-functional umuDC gene.
Polypeptide expression and collection
A method for producing a polypeptide by incubating the bacterium under conditions suitable for expressing the polypeptide and collecting the polypeptide.
Overall, the claims cover an engineered reduced-genome E. coli bacterium characterized by specified insertion sequence deletions, functional absence of polB and dinB, optionally umuDC, and deletion of defined DNA segments from MG1655. Dependent claims further narrow the native parent strain to MG1655, specify a non-functional umuDC gene, and include steps for expressing and collecting an encoded polypeptide.
Stated Advantages
Improved genetic stability.
Very low mutation rate.
Reduced spontaneous mutation rates.
Reduced mutation-rate response under stresses including mitomycin C and protein overexpression.
Delayed accumulation of loss-of-function mutations in a toxic gene carried on a plasmid.
Documented Applications
Using the engineered bacterium to carry and maintain a toxic gene on a plasmid (SinI/SinI methyltransferase system) with delayed accumulation of loss-of-function mutations.
Producing and collecting a polypeptide expressed by the engineered bacterium under conditions suitable for expressing the polypeptide.
Assessing mutation rates using cycA mutation assays (fluctuation analysis) and other assays including stress conditions such as mitomycin C and protein overexpression.
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