阅读说明：本技术 通过调控突变率的定向进化 (Directed evolution by modulating mutation rates ) 是由 彼得·B·雷因茨 于 2017-12-20 设计创作，主要内容包括：本发明涉及用于大分子定向进化的方法和系统。所述方法涉及增加包含编码缺乏期望活性的基因产物的感兴趣基因的进化生物的突变率,由此产生编码包含期望活性并引起诱变抑制的进化基因产物的突变的感兴趣基因。所述系统包括包含编码待进化基因产物的感兴趣基因的进化生物、宿主生物以及任选的进化池、cellstat和/或合适的生长培养基。(The present invention relates to methods and systems for directed evolution of macromolecules. The methods involve increasing the mutation rate of an evolved organism comprising a gene of interest encoding a gene product lacking a desired activity, thereby generating a gene of interest encoding a mutation of the evolved gene product comprising the desired activity and causing mutagenesis inhibition. The system comprises an evolving organism comprising a gene of interest encoding a gene product to be evolved, a host organism and optionally an evolution cell, a cell and/or a suitable growth medium.)

1. A method of directed evolution of a macromolecule, the method comprising artificially increasing the mutation rate experienced by a population of an evolving organism comprising a gene of interest to be evolved, wherein the gene of interest encodes a gene product that does not comprise a desired activity, thereby causing the gene of interest to evolve in one or more evolving organisms, thereby encoding an evolved gene product comprising a desired activity, wherein the mutation rate is proportionally reduced in response to the production of the evolved gene product comprising the desired activity, thereby facilitating propagation of the one or more evolving organisms comprising the evolved gene of interest in the population and at a reduced mutation rate.

2. the method of claim 1, wherein the evolved organism requires a host organism for replication.

3. The method of claim 2, wherein the host organism is engineered to have an increased mutation rate relative to the mutation rate of a host organism that is not engineered to have an increased mutation rate.

4. The method of claim 2 or 3, wherein artificially increasing the mutation rate experienced by the evolving biological population comprises contacting the evolving biological population with the host biological population, wherein the contacting optionally occurs in an evolution pond.

5. The method of claim 3 or 4, wherein the engineered host organism comprises a plasmid or modified host chromosome containing a set of inducible genes encoding one or more mutagenic components.

6. The method of claim 5, wherein the one or more mutagenic components are selected from the group consisting of a modified DNA polymerase protein domain capable of reducing the fidelity of DNA replication by a DNA polymerase in the host organism, and a protein capable of interacting with a DNA polymerase in the host organism to reduce the fidelity of DNA replication by the DNA polymerase.

7. The method of claim 5 or 6, wherein increasing the mutation rate comprises introducing a plasmid containing a phage shock promoter capable of inducing the generation of the mutagenized component.

8. The method of any one of claims 5-7, wherein the mutation rate is reduced by generating and down-regulating repressor molecules for expression of the mutagenic component in proportion to the generation of the desired activity.

9. A method according to claim 8, wherein the repressor is a tetracycline repressor (TetR) and the plasmid comprises a TetR binding site comprising one or more repeats of a tetracycline operator sequence or a tetracycline responsive element.

10. The method of any one of claims 1-10, wherein mutagenesis is increased by introducing a mutagenic compound into the environment of the evolved organism.

11. The method of claim 1, wherein the evolved organism is a modified M13 bacteriophage, wherein the genome of the evolved organism comprises a subset of wild-type M13 bacteriophage genes comprising the DNA sequences of gii, gIV and the origin of replication and the DNA sequences of a packaging signal, wherein all other M13 bacteriophage genes essential for bacteriophage propagation are encoded in a plasmid comprised by the host organism and are constitutively expressed as a response to bacteriophage infection or are induced to be expressed by an external agent.

12. The method of claim 11, wherein the host organism is Escherichia coli (Escherichia coli).

13. The method according to any one of claims 2-11, wherein the evolved organism is a modified M13 bacteriophage, wherein the genome of the evolved organism comprises a subset of wild-type M13 bacteriophage genes, said subset comprising the DNA sequences of gii, gIV and the origin of replication and the DNA sequences of the packaging signal, wherein all other M13 bacteriophage genes necessary for bacteriophage propagation are encoded in a plasmid in the host organism and are constitutively expressed as a response to bacteriophage infection or are induced to expression by an external agent.

14. The method of claim 13, wherein the host organism is escherichia coli.

15. A system for directed evolution of macromolecules, the system comprising:

(a) A population of evolved organisms comprising a gene of interest to be evolved, wherein the gene of interest encodes a gene product that does not comprise a desired activity, thereby enabling the gene of interest to evolve in one or more evolved organisms that require replication by a host organism, thereby encoding an evolved gene product comprising a desired activity and the ability to directly or indirectly inhibit induced mutagenesis; and

(b) A population of host organisms, wherein the host organisms are engineered to have an increased mutation rate compared to the mutation rate of host organisms that are not engineered to have an increased mutation rate.

16. The system of claim 15, further comprising at least one member selected from the group consisting of: a container, a cellstat, and a growth medium suitable for propagating the evolved organism and/or the host organism.

17. The system of claim 15 or 16, wherein the evolved organism is a bacteriophage and the host organism is a bacterium.

18. The system of claim 17, wherein the bacteriophage is M13 and the host organism is e.

19. The system of claim 18, wherein the M13 bacteriophage is a modified M13 bacteriophage comprising a subset of wild-type M13 bacteriophage genes comprising the DNA sequences of gii, gIV, and origin of replication and packaging signals, wherein all other M13 bacteriophage genes necessary for bacteriophage propagation are encoded in a plasmid comprised by the host organism and are constitutively expressed in response to bacteriophage infection or are induced to be expressed by an external agent.

20. The system of claim 15 or 16, wherein the evolved organism is the evolved organism of any one of claims 1-14.

21. The system of any one of claims 15, 16 and 20, wherein the host organism is the host organism of any one of claims 2-14.

22. A gene product comprising a desired activity produced by the method of any one of claims 1-14.

23. The gene product of claim 22, wherein the gene product is a protein or RNA.

24. A nucleic acid molecule encoding the gene product of claim 22 or 23.

25. A nucleic acid molecule encoding a gene product comprising a desired activity, the nucleic acid molecule produced by the method of any one of claims 1-14.

26. a cell or virus comprising a gene product according to claim 22 or 23 and/or a nucleic acid molecule according to claim 24 or 25.

27. The cell of claim 26, wherein the cell is selected from the group consisting of a bacterial cell, an animal cell, a mammalian cell, a human cell, a fungal cell, an algal cell, and a plant cell.

28. The cell of claim 26, wherein the cell is a non-human cell or a human cell cultured in vitro.

Technical Field

The present invention relates to the field of directed evolution of nucleic acids and gene products encoded thereby.

Background

The continuous evolution of bacteriophages has the potential to become a powerful tool for protein engineering. In this process, the continuous evolution of microorganisms rapidly generates DNA sequences encoding proteins which undergo multiple generations of mutation and select for specific properties of said proteins. The versatility of this protein engineering approach is limited by our ability to insert expressible initial genes into replicating microorganisms and create a selection mechanism for the desired activity.

Currently, the best practice of directed evolution has been to exploit the rapid propagation of the bacterial virus M13 filamentous bacteriophage to evolve new proteins. Transformed e.coli (e.coli) host cells provide high levels of mutation and a fitness selection mechanism that rewards virus particles that exhibit desirable properties. As with natural evolution, directed evolution requires repeated cycles of the following three processes: (1) variation or mutation to allow for new or enhanced functionality; (2) selecting to confer reproductive advantage to an individual having a desired new or enhanced function relative to an individual exhibiting a lesser degree of said function; (3) propagate to pass the selected function to the next generation.

Current continuous evolution programs induce elevated mutation rates with external agents in order to rapidly sample most of the evolution landscape (evolution landscapes). These elevated mutation rates must be avoided during host cell culture and can be induced by external agents when the host cells have been transferred to an environment where they are infected by the evolving organism. As a result, both the replicating host cell and the resulting virion may contain a large number of mutations.

Engineering new proteins by continuous evolution of bacteriophages currently requires transformation of host bacteria with two additional functions: (1) mutagenic vectors that provide increased viral mutation rates, and (2) selection mechanisms that provide reproductive advantages to genotypes encoding products that produce desired activities.

A technique called Phage Assisted Continuous Evolution (PACE) creates a fast-evolving environment by inducing high levels of mutagenesis in phage-containing vessels (U.S. patent No. 9,394,537). Because the mutagenesis is externally induced, it affects all entities within the container. In particular, even uninfected E.coli cells undergo enhanced levels of mutation when they grow and divide. The effect caused by the presence of E.coli mutants is mitigated by carefully controlled flow rates which in fact ensure that a given E.coli cell is in a vessel containing the evolved phage for less than one life cycle (disruption time). One to four volumes per hour is sufficient to ensure that less than one percent of the E.coli remains in the vessel for as long as it is sufficient to divide and produce daughter cells containing the mutation.

Specifically, the PACE system requires: (1) a modified viral genome, replacing a critical phage gene with a gene to be evolved, (2) a transformed host with inducible mutagenesis, (3) a host plasmid containing a selection mechanism to provide the critical phage gene proportional to the desired activity of the evolved gene, and (4) a transit time of e.

The PACE process removes a key gene from the M13 phage and replaces it with the protein to be evolved, thereby making the evolved protein a replacement for the protein desired by the phage. The selection mechanism requires that the host cell produce or inhibit this key phage protein in order to reward or clear the phage encoding the protein of interest.

This approach creates difficulties in at least two ways. First, the removal of key genes from phages results in incomplete phages that are not readily propagated prior to the evolution experiment. The propagation of phages requires the manual supply of key proteins. PACE includes additional mechanisms that allow for the external induction of key protein production.

The second difficulty relates to the key phage gene selection currently employed for PACE implementation: fusion proteins (pIII). The presence of this protein in E.coli is usually the result of infection with M13, which elicits a response that prevents subsequent phage infection. This infection response is one of the reasons that M13-infected e.coli is an efficient platform for directed evolution. Coli responds and rewards the first M13 to infect it, producing only the phage progeny that are progeny of that phage. If superinfection is possible, E.coli will produce progeny of multiple phage lineages, the selection mechanism of which is influenced by the combination of their evolved proteins.

The host mechanism for producing pIII fusion proteins must not only be externally inducible in order to propagate the defective phage, but also be tightly controlled until infection occurs. This is because the inadvertent production of pIII in the host cell elicits an infectious response that prevents any phage infection.

Current directed evolution methods induce mutagenesis by introducing mutagenic compounds into the culture medium of a container containing an evolved phage population, thereby imparting an overall constant mutation rate to the phage population. The mutagenic compound may be a small molecule that directly causes a mutation, or triggers an inducer that produces an error-prone polymerase component. Using this second approach, cells producing varying amounts of modified polymerase subunits will have a corresponding DNA replication error rate. The ability to activate the expression of these mutagenized components in the host cell makes this method possible.

Disclosure of Invention

The method of the invention realizes a continuous evolution system using mutagenesis inhibition as a selection mechanism. The desired activity of the evolved macromolecule results in a higher number of progeny that are identical to the parent, rewarding the genotype by increasing the number of phage carrying the genotype.

The methods of the invention involve a host cell culture system that provides uninfected E.coli cells to a container containing an evolved phage population. The phage contains the complete wild-type genome plus the genes for the protein to be evolved. As in other methods of continuous evolution, mutagenesis is induced in a host cell, either in its entirety or in response to infection. However, the selection mechanism consists of: the mutation rate is reduced in response to the desired activity of the evolved protein. Individuals containing DNA sequences encoding proteins with the desired activity will acquire a lower mutation rate when replicated, producing more progeny with the same genotype as the parent phage. We refer to this method of selection as mutagenic selection or M-selection.

Phages encoding evolved proteins that do not exhibit the desired activity will continue to receive the maximum induced mutation rate. Although this selection mechanism does not immediately eliminate ill-behaved individuals, their lineage will experience extinction due to an error catastrophe (error catastrophe) within generations. Of course, unless one of the mutants expresses an improvement in the evolved protein, in this case its progeny will start to undergo a reduced mutation rate and thus a more faithful reproduction.

The method of the invention has the following characteristics: lineages with an increased level of desired activity will be less actively searched for evolutionary landscape, while poorly performing individuals continue to actively explore the mutation space with the risk that their lineages experience error disasters.

In one embodiment of the invention, the method described herein also employs an E.coli infection responsive phage shock promoter as a means of inducing increased mutagenesis. Thus, uninfected host cells are not subjected to enhanced mutations and can stay indefinitely in the evolutionary pool (lagon) for infection. Infected cells grow slower than uninfected cells because energy is put into phage production, which slows down the cell life cycle and reduces the need for high flow rates. Furthermore, infected cells produce only pre-infected daughter cells, which eliminate any interaction between the mutated E.coli and progeny phage.

The mutagenesis selection method reduces the difficulty of breeding incomplete phages. The protein to be evolved is added to fully functional phage as in classical phage display. Mutagenic selection also avoids the problems associated with unwanted pIII production in host cells by eliminating the need for host plasmids encoding the pIII gene.

An important difference between PACE and the present disclosure is that the mutagenesis is not externally induced, and therefore, uninfected cells are not mutated, enabling the production of a minimal number of faithful copies before the mutagenized product is expressed by the infected host. This may confirm that preserving the arabinose-induced mutagenesis of PACE is useful as a way to achieve moderate baseline mutation rates in the evolutionary pool.

This lacks externally induced mutagenesis, and thus mutagenesis in uninfected cells alleviates the limitation on the passage time of the evolutionary pool of a host cell life cycle. In addition, M13 infection slows down the doubling time of E.coli, further alleviating this limitation. If uninfected cells propagate in the evolutionary pool they will not mutate, if infected cells propagate, daughter cells will be pre-infected and will simply continue to remove the virus infecting the parent at the level of the mutation determined by the original infection. Thus, a wider range of flow rates can be used in mutagenic selection.

Another important difference between the method of the present invention and PACE is that the mutation space is less aggressively searched when an improvement in activity is desired. Genotypes that experience an increase in activity do not search the mutation space as aggressively, while mutants with little or no activity continue to search the mutation space aggressively. The use of such mixed populations and weak selection delays the fixing of genotypes, allows the use of extended evolutionary landscape, and helps to ensure that the system does not fall into local fitness peaks.

Because the key genes are not removed from the phage, it can be readily propagated before the directed evolution process is initiated. The problems associated with pIII present in the host and the associated premature infection response are avoided because no phage genes are incorporated into the host plasmid. Additional negative selection mechanisms can be added by blocking repressor molecules or expressing additional mutagenic components separately.

Drawings

FIG. 1 shows a general schematic of mutagenic selection (M selection).

FIG. 2 shows an example of M selection using a phage shock promoter and a Tet repressor, where two-hybrid protein-protein binding causes expression of the tetracycline repressor gene (tetR) resulting in the tetracycline repressor protein (TetR) that is capable of binding to a tetracycline responsive element or other tetracycline operator sequence in the plasmid or modified host chromosome, thereby blocking transcription of the phage shock promoter.

FIG. 3 shows an example of a response to a bacteriophage exhibiting a low level of desired activity. Weak binding allows higher mutation rates.

Figure 4 shows an example of a response to a bacteriophage exhibiting a high level of desired activity. Strong binding produced more clones.

Figure 5 shows an example of M-selection to evolve T7 polymerase to recognize or bind the T3 polymerase promoter.

Detailed Description

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Definition of

"desired activity" is a property of an evolved gene product (e.g., a protein or RNA) that we wish to enhance. For example, the desired activity may be the ability to bind to a particular nucleotide sequence in another protein or nucleic acid molecule designated as a target (e.g., dsDNA, ssDNA, ssRNA, dsRNA, etc.). The desired activity may also be an enzymatic activity. It is recognized that the initial minimum level or amount of desired activity can be further enhanced using the methods of the present invention. For example, the desired activity may be the binding of an evolved gene product to a particular protein. Initially, the desired activity may be the minimum level of binding of the gene product to a particular protein sufficient to cause minimal mutagenesis inhibition. However, using the methods of the invention, the level of binding of a gene product to a particular protein can be further increased or enhanced, thereby further enhancing the suppression of mutagenesis.

"target" is the protein or nucleic acid substrate that we are trying to affect. The target is produced by the host organism and is not subject to evolutionary pressure. This may be, for example, a protein or nucleic acid substrate, particularly a particular nucleotide sequence in which we wish our evolved protein to bind.

A "host organism" is a microorganism transformed with one or more nucleic acid molecules, wherein the transformed microorganism is capable of affecting the propagation of the evolved organism based on the desired activity exhibited by the evolved or evolved gene product. Preferably, the microorganisms should not be affected by the pressure of evolution.

A "selection mechanism" is a process by which a particular desired activity results in increased reproduction of the entity encoding the gene associated with that activity. For example, a protein carried by a bacteriophage that is subject to an increase in mutation rate is selected based on the extent to which a second protein that is not subject to evolutionary pressure is bound.

"cellstat" is a container in which host cells are propagated prior to infection by an evolved organism (e.g., a bacteriophage). The cellstat environment is designed to maintain low levels of mutagenesis and avoid selective pressure on host cells (e.g., bacterial cells, e.

An "evolutionary pool (lagon)" is a container in which a population of host organisms (e.g., bacteria) is in contact with and can be infected by a population of evolved organisms (e.g., bacteriophage) and in which the progeny of the evolved organisms are maintained. The evolutionary pool environment is designed to enhance mutagenesis in order to accelerate the evolution of the gene of interest and the gene product encoded thereby.

"error catastrophe" is the extinction caused by the accumulation of excessive mutations.

By "weakly selected" is meant that the organism is unable to settle in the evolving population because its fitness is only slightly higher than that of its competitors.

"population" is intended to mean two or more individuals, but generally speaking, a population of the invention will contain 104, 105, 106 or more individuals (e.g., bacteriophage, bacteria), it being understood that the number of individuals in a population of the invention may vary depending on any number of factors, including, for example, the volume of the evolutionary pool or other container, the species of the evolved organism, the species of the host organism, and environmental conditions.

An "evolving organism" (evolved organism) is an organism to be evolved which comprises a gene of interest encoding a gene product of interest. Preferred evolved organisms of the invention are viruses, in particular bacteriophages, more particularly M13.

"superinfection" refers to the infection of a single host cell by more than one virus.

Description of the invention

Now, very high but still controllable mutation rates in vivo are possible. This broad-spectrum mutagenesis has up to 105-fold basal mutation rates and can be controlled within a single host cell. If this mutagenesis is initiated by infection, such as with a phage shock promoter, but is subsequently reduced by the desired activity, then two evolutionary major components will be met. An increase in the number of identical phage progeny produced by an individual exhibiting the desired activity will select for the desired activity. This is the mutagenic selection. While previous methods induced uniform mutagenesis in the evolutionary pool, mutagenic selection induced a unique level of mutagenesis in each infected host cell.

Although clone and mutant might sound like a bad science fiction movie title, it just illustrates how the mutagenic selection works. This process results in more mutants if the genotype does not encode a protein with the desired properties, and more clones if the genotype results in the expression of a protein with the desired properties. The level of mutagenesis is adjusted between maximum mutagenesis in the presence of a protein that does not exhibit the desired activity and very low levels of mutagenesis in the production of a protein that exhibits the desired activity.

It is necessary to produce two clones per generation to infect the host to produce enough phage to prevent genotype extinction (washout). Assuming phage production of 100/hr and a cell passage time of 1 hr, the maximum mutation rate of four (4) mutations per genome will avoid disappearance. The percentage of phage progeny that are exact copies of the parent is derived from a poisson distribution, where μ ═ 0 is the expected number of mutations and λ is the mutation rate per virion (mutation rate/base 6.4 kbp/genome). The mutation rate of λ ═ 4 gave a zero mutation probability of 1.8%.

The genome is therefore able to tolerate and have a maximum mutation rate per base left by any clone in the evolutionary pool of 4/6400 ═ 6.25X 10-4. The basal rate of E.coli producing M13 phage was 7.2X 10-7. The average production of normal M13-infected phage was 100/hr and assuming a passage time of 1 hour for the host cell, a minimum cloning efficiency of 2% was required: two faithful copies were infected at a time to avoid disappearance. Previous high mutation rate considerations seem to allow slower flow rates, which would lower the minimum fraction.

FIG. 1 shows an overview of the mutagenic selection process. The inducer activates the mutagenesis by allowing the mutagenic component to be expressed. When the desired activity is produced, a repressor protein is expressed and expression of the mutagenized component is inhibited. The amount of mutagenesis inhibition is proportional to the amount of desired activity. The reduction in mutagenesis increases the number of non-mutated phage progeny.

Figure 2 shows an example of how this might work. The interaction between the target protein (constitutively expressed by the host organism and not subject to evolutionary stress) and the evolved protein encoded by the phage and expressed in the infected host causes expression of the repressor, which reduces the expression of the mutagenized component. The increased binding affinity between the target and the evolved protein results in a higher level of repressor expression and thus a lower mutation rate in the host cell. The phage shock promoter is a particularly good inducer to activate nominal mutation levels, ensuring that only infected cells will have higher levels of mutation. Since the phage-shock promoter is activated by the p4 protein (pIV) expressed after infection and the repressor is activated by the expression of the evolved protein, the activator and repressor components will compete simultaneously to regulate the mutation rate.

Figure 3 shows an example of a response to a phage encoding an evolved protein with a low level of the desired behavior, in this case binding to a target protein. Because repressors are expressed with little or no expression, the mutation rate remains high as determined by response to the phage shock promoter. Progeny produced by the host organism will contain a large number of mutants and therefore actively search for an evolutionary landscape, with few clones or exact copies of this poorly expressing phage genotype.

FIG. 4 shows an example of a response to a bacteriophage encoding a protein with a high level of desired activity. The strong binding between the evolved protein and the target results in enhanced repressor expression, resulting in lower yields of mutagenic components and thus lower mutation rates. Fewer of the resulting progeny will contain mutations, resulting in a less aggressive search for variations, and more will carry an exact copy of the genotype encoding a protein with a high level of the desired activity.

Non-limiting embodiments of the invention include, for example, the following embodiments.

1. A method of directed evolution of a macromolecule, the method comprising artificially increasing the mutation rate experienced by a population of an evolving organism containing a gene of interest to be evolved, wherein the gene of interest encodes a gene product that does not contain a desired activity, thereby causing the gene of interest to evolve in one or more of the evolving organisms, thereby encoding an evolved gene product containing the desired activity, wherein the mutation rate is proportionally reduced in response to the production of the evolved gene product containing the desired activity, thereby facilitating propagation of the one or more evolved organisms containing the evolved gene of interest in the population and at the reduced mutation rate.

2. The method of embodiment 1, wherein the evolving organism requires the host organism to replicate.

3. The method of embodiment 2, wherein the host organism is engineered to have an increased mutation rate relative to the mutation rate of a host organism that is not engineered to have an increased mutation rate.

4. The method of embodiment 2 or 3, wherein artificially increasing the mutation rate experienced by the evolving biological population comprises contacting the evolving biological population with a host biological population, wherein the contacting optionally occurs in an evolution pond.

5. The method of embodiment 3 or 4, wherein the engineered host organism comprises a plasmid or modified host chromosome containing a set of inducible genes encoding one or more mutagenic components.

6. The method of embodiment 5, wherein the one or more mutagenic components are selected from the group consisting of modified DNA polymerase protein domains capable of reducing the fidelity of DNA replication by a DNA polymerase in the host organism, and proteins capable of interacting with a DNA polymerase in the host organism to reduce the fidelity of DNA replication by the DNA polymerase.

7. The method of embodiment 5 or 6, wherein increasing the mutation rate comprises introducing a plasmid containing a phage shock promoter capable of inducing the production of the mutagenized component.

8. The method of any of embodiments 5-7, wherein the mutation rate is reduced by generating and down-regulating repressor molecules expressed by the mutagenic component in proportion to the generation of the desired activity.

9. The method of embodiment 8, wherein the repressor is a tetracycline repressor (TetR) and the plasmid comprises a TetR binding site comprising one or more repeats of a tetracycline operator sequence or a tetracycline responsive element.

10. The method of any one of embodiments 1-10, wherein the mutagenesis is increased by introducing a mutagenic compound into the environment of the evolved organism.

11. The method of embodiment 1, wherein the evolved organism is a modified M13 bacteriophage, wherein the genome of said evolved organism comprises a subset of wild-type M13 bacteriophage genes comprising the DNA sequences of gii, gIV and the origin of replication and the DNA sequences of the packaging signal, wherein all other M13 bacteriophage genes essential for bacteriophage propagation are encoded in a plasmid comprised by the host organism and are constitutively expressed as a response to bacteriophage infection or are induced to be expressed by an external agent.

12. The method of embodiment 11, wherein the host organism is Escherichia coli (Escherichia coli).

13. The method of any one of embodiments 2-11, wherein the evolved organism is a modified M13 bacteriophage, wherein the genome of said evolved organism comprises a subset of wild-type M13 bacteriophage genes comprising the DNA sequences of gii, gIV and the origin of replication and the DNA sequences of the packaging signal, wherein all other M13 bacteriophage genes necessary for bacteriophage propagation are encoded in a plasmid in the host organism and are constitutively expressed as a response to bacteriophage infection or are induced to be expressed by an external agent.

14. The method of embodiment 13, wherein the host organism is escherichia coli.

15. A system for directed evolution of macromolecules, the system comprising:

(a) A population of evolved organisms comprising a gene of interest to be evolved, wherein said gene of interest encodes a gene product that does not comprise a desired activity, thereby enabling said gene of interest to evolve in one or more evolved organisms, thereby encoding an evolved gene product comprising a desired activity and the ability to directly or indirectly inhibit induced mutagenesis, wherein the evolved organisms require replication by a host organism; and (b) a population of host organisms, wherein the host organisms are engineered to have an increased mutation rate compared to the mutation rate of host organisms that are not engineered to have an increased mutation rate.

16. the system of embodiment 15, further comprising at least one member selected from the group consisting of: a container, a cellstat, and a growth medium suitable for propagating the evolved organism and/or the host organism.

17. The system of embodiment 15 or 16, wherein the evolved organism is a bacteriophage and the host organism is a bacterium.

18. The system of embodiment 17, wherein said bacteriophage is M13 and said host organism is e.

19. The system of embodiment 18, wherein the M13 bacteriophage is a modified M13 bacteriophage comprising a subset of wild-type M13 bacteriophage genes comprising the DNA sequences of gii, gIV, and an origin of replication and a packaging signal, wherein all other M13 bacteriophage genes necessary for bacteriophage propagation are encoded in a plasmid comprised by the host organism and are constitutively expressed as a response to bacteriophage infection or are induced to be expressed by an external agent.

20. The system of embodiment 15 or 16, wherein the evolved organism is the evolved organism of any one of embodiments 1-14.

21. The system of any one of embodiments 15, 16 and 20, wherein the host organism is the host organism of any one of embodiments 2-14.

22. A gene product comprising a desired activity produced by the method of any one of embodiments 1-14.

23. The gene product of embodiment 22, wherein the gene product is a protein or RNA.

24. A nucleic acid molecule encoding the gene product of embodiment 22 or 23.

25. A nucleic acid molecule encoding a gene product comprising a desired activity, the nucleic acid molecule produced by the method of any one of claims 1-14.

26. A cell or virus comprising the gene product of embodiment 22 or 23 and/or the nucleic acid molecule of embodiment 24 or 25.

27. The cell of embodiment 26, wherein said cell is selected from the group consisting of a bacterial cell, an animal cell, a mammalian cell, a human cell, a fungal cell, an algal cell, and a plant cell.

28. The cell of embodiment 26, wherein said cell is a non-human cell or a human cell cultured in vitro.

Alternative embodiments

Other mechanisms may be employed to reduce mutation rates in response to a desired activity, including but not limited to RNA interference with mRNA encoding a mutagenized polymerase component, stimulation of production of a highly accurate polymerase component, or activation of an anti-mutant gene.

With the development of new methods for inducing high mutation rates, new methods will undoubtedly be proposed to regulate the resulting mutation rates. Thus, a clear extension of this approach is the use of this regulation as a selection mechanism in continuous evolution.

The articles "a" and "an" are used herein to mean either one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" means one or more elements.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Where reference is made herein to a method comprising two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes that possibility), the method may comprise one or more further steps performed before any defined step, between two defined steps or after all defined steps (unless the context excludes that possibility).

The term "at least" followed by a number is used herein to indicate that the beginning of a range includes the number (which may be a range with or without an upper limit, depending on the variable being defined). For example, "at least 1" means 1 or greater than 1. The term "at most" followed by a number is used herein to indicate that the end of a range includes the number (which can be a range having 1 or 0 as its lower limit or a range having no lower limit, depending on the variable being defined). For example, "at most 4" means 4 or less than 4, "at most 40%" means "40% or less than 40%". In this specification, when a range is given as "(first digit) to (second digit)" or "(first digit) - (second digit)", this means a range including two digits as a boundary. For example, "25 to 100" means a range having a lower limit of 25 and an upper limit of 100, and includes both 25 and 100.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.