Directed evolution of microorganisms

Abstract

The present invention provides methods for directing the evolution of microorganisms comprising the use of mutator genes and growth under conditions of selective pressure. The method discloses mutator genes which can be used in the methods of the present invention and provides ATCC deposits which exemplify the evolved microorganisms produced by the methods.

Description

FIELD OF THE INVENTION[0001]The present invention relates to methods for directing the evolution of microorganisms using mutator genes. Such methods provide a pool of microbial genetic diversity advantageous for industrial application, such as for the industrial production of heterologous proteins, such as hormones, growth factors and enzymes, and the biocatalytic production of chemicals, vitamins, amino acids and dyes.BACKGROUND OF THE INVENTION[0002]The industrial applicability of microorganisms is restricted by their physiological limits set by solvent, pH, various solutes, salts and temperature. Organic solvents are generally toxic to microorganisms even at low concentrations. The toxicity of solvents significantly limits use of microorganisms in industrial biotechnology for production of specialty chemicals and for bioremediation applications. Solvent molecules incorporate into bacterial membranes and disrupt membrane structure (Isken and Bont, 1998, Extremophiles 2(3): 229-238...

AI Technical Summary

Benefits of technology

[0034]Additionally, the methods of the present invention provide an advantage for obtaining microorganisms comprising desired phenotypic traits associated with multiple genes, such as the ability of a microorganism to grow at elevated temperatures. The use of the mutator gene provides a means for producing genetic diversity and the simultaneous growth under conditions of selective pressure allows the microorganism to identify the specific genetic changes required for survival. The use of mutD gene mutations allows for very large diversity to be provided to the microorganism from which to select for the specific genetic changes that provide a growth advantage. Therefore, the methods disclosed herein avoid the limited diversity that is often produced with art methods that begin the directed evolution process with defined sets of genes. Furthermore, the methods disclosed herein eliminate additional screening steps often associated with art methods for producing genetic diversity. A further advantage of the present invention is that the methods can be applied to microorganisms which have not been sequenced and for which there may be limited information upon which to design genetic changes.

[0035]In illustrative embodiments disclosed herein, a mutated mutD gene residing on a plasmid was introduced via recombinant techniques into E. coli or E. blatte. The E. coli or E. blatte cell was then cultured under conditions suitable for growth for a time sufficient for at least 20 doublings and up to at least about 2000 doublings under conditions of selective pressure. In one example, E. coli was grown under conditions of increased temperature or in the presence of DMF and in another E. blattae was growth in the presence of solvent, such as DMF or 1,3 propanediol. As a result, E. coli was evolved into a microorganism capable of growing at temperatures up to about 48° C. or in the presence of 80 g / l DMF. E. coli evolved to grow at elevated temperatures also became auxotrophic for three amino acids, Cys / Met, Asp / Asn and Pro. E. blattae was evolved into a microorganism capable of growing anaerobically in the presence of at least 105 g / l 1,3-propanediol and which comprised genetic changes in at least one catalytic activity associated with 1,3 propanediol production, 1,3-propanediol dehydrogenase, shown in FIG. 3.

[0036]The use of a plasmid comprising a mutator gene, ie, a mutator plasmid, can be used to control the mutation rate of a microorganism. As described under Section II below, plasmid constructs can be designed which provide reduced levels of expression of a mutator gene thereby providing a means for altering the ratio of naturally occurring DNA repair genes vs mutator genes. This provides a means for combining the advantage of mutD mutations (which results in random mutagenesis) with the advantages of the other known mutators (lower mutation frequency which leads to a lower burden on the cells). Additionally, plasmid constructs can be designed that allow for curing the evolved microorganism of the mutator gene, such as the use of a temperature sensitive origin, thereby allowing for a means for turning the mutation events off and on in the microorganism. For a gram positive microorganism, such as B. subtilis where the entire genome has been sequenced, the present invention could encompass the steps of deleting or mutating a DNA repair gene, evolving the Bacillus, and restoring the naturally occurring DNA repair system through recombination events. As disclosed herein, several members of Escherichia, such as E. coli and E. blatte have been subjected to the directed evolution methods. Illustrative examples of evolved E. coli and E. blattae have been deposited with the ATCC and have accession numbers, ______ and ______, respectively.

[0037]The methods of the present invention provide a means to accomplish long-term evolution of microorganisms. An E. coli strain comprising a plasmid comprising a mutD mutation was grown for >1000 doublings without a reduction in mutation rate. The present invention also provides a means for reducing the functional genome of an organism. A microorganism can be grown for many thousands of generations, such that only the genes which are essential would remain functional. Most of the other genes would carry random and inactivating mutations.

[0038]The present invention also provides a means for making non-pathogenic organisms. A pathogenic strain can be evolved into a mutator strain by introduction of a mutator gene and grown for extended periods of time. As a result many of the genes that are involved in pathogenicity would become inactivated and the strain would be safe to use.

[0039]The present invention also provides a means to streamline the metabolism of an organism. A strain which has an improved yield on nutrients or a reduced metabolic rate (maintenance metabolism) can be produced by methods disclosed herein. Such strains would be useful production strains for chemicals as well as enzymes. The present invention provides a means for making microorganisms mutator strains by introducing a mutator gene, thereby protecting the microorganism's naturally occurring DNA repair genes from becoming mutator genes in response to selective pressure. That is, the introduction of the mutator plasmid into a microorganism whether via a plasmid or into the genome, protects the cells from developing a mutator phenotype in response to selective pressure.

Problems solved by technology

Organic solvents are generally toxic to microorganisms even at low concentrations.

The toxicity of solvents significantly limits use of microorganisms in industrial biotechnology for production of specialty chemicals and for bioremediation applications.

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