Patent Publication Number: US-10760071-B2

Title: System for continuous mutagenesis in vivo to facilitate directed evolution

Description:
RELATIONSHIP TO OTHER APPLICATIONS 
     This application claims benefit of and priority to U.S. Ser. No. 62/196,527 titled System for continuous mutagenesis in vivo to facilitate directed evolution, filed 24 Jul. 2015 which is incorporated by reference for all purposes. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with government support under NSF grant No. S0183542. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been filed electronically in ASCII form and is hereby incorporated by reference in its entirety. Said ASCII copy, created May 4, 2020, is named 102913-001300US-1070528_SL.txt and is 127,584 bytes in size. 
     BACKGROUND 
     This invention overcomes a limitation of in vivo mutagenesis systems, which is that mutagenesis rapidly declines with continuous culture, precluding the simultaneous implementation of mutagenesis and selection. Concurrent mutagenesis and selection is the most efficient way to conduct evolution in the laboratory. The alternative is iterative rounds of directed evolution, i.e. to generate random mutant libraries separately, and select them once they have been created. 
     BRIEF DESCRIPTION OF THE INVENTION 
     This system of random mutagenesis performed in vivo is based on expressing an error-prone allele of Pol I (EP-Pol I, bearing three mutations: I709N, A759R, and D424A) in a polA12 (Pol A is strain) of  E. coli  and placing a gene of interest in a ColE1 plasmid. When the ColE1 plasmid is replicated by EP I, random mutations are introduced. Alternative methods of mutagenesis in vivo include replication of a plasmid in an error-prone Pol III strain of  E. coli , and replication in mismatch repair-deficient strains, although in these cases mutagenesis is not stable either. Plasmids can be treated with mutagenic chemicals or UV prior to transformation, but these methods produce highly biased mutation spectra. Error-prone replication of phage represents an alternative to error-prone plasmid replication and is ideal for evolution of surface proteins, an approach known as phage display. 
     The inventors found that the decline in mutagenesis in culture is clonal, i.e., that by performing the experiments in parallel with several clones, the inventor found cell lines showing continuous mutagenesis. This is detected with the help of a fluorescent reporter of mutagenesis. 
     In addition, the inventors have identified a novel mutant polymerase with a missense mutation at position 54 of DNA polymerase I (K54E) that enhances continuous mutagenesis. This mutation was identified by selection of a random mutant library of Pol I for efficient long-term mutagenesis in vivo and has never been described before. It is located in the p5′ to 3′ exonuclease domain of the polymerase and has never been described before. 
     Pol I is a family A polymerase, which includes a variety prokaryotic and organel DNA polymerases specialized in gap repair and replication of circle DNA. Pol I has a polymerase and a 5′ to 3′ exodomain. Both domains are fused but functionally independent. Prokaryotic 5′ to 3′ exonucleases share significant sequence homology with the polymerase-independent 5′ nucleases from several bacteriophages and are also related to mammalian FEN-I proteins to proteins of the RAD2 family. Six conserved regions were identified through the multiple sequence alignment of the bacterial and bacteriophage nucleases. See  FIG. 1 . The relevant K residue in  E. coli  Pol I (top sequence of  FIG. 1 ) is highlighted with an arrow, and is located four amino acids 5′ to conserved motif B. Note that both Tq ( Thermus aquaticus ) and Tf ( Thermus flavus ) have acidic residues at this position (D), similar to our mutation (E). Tq Polymerase is the polymerase routinely used for PCR. See  FIG. 1 . 
     
       
         
           
               
             
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     Thus the invention consists of using a DNA polymerase carrying the following point mutations K54E, I709N, A759R, D424A (which henceforth is called “K54E LF Pol I” and this method is believed to be novel) and setting up multiple lines. K54E is believed to be an entirely novel mutation, the other three mutations (I709N, A759R, D424) were previously described in a paper authored by the inventor. 
     Using a GFP reporter present in the ColE1 plasmid, the inventors can detect lines where mutagenesis is continuous and does not exhibit the usual decline in mutagenesis with sequential cloning. Continuous mutagenesis is detectable by sequencing a set of plasmids following sequential passage or using other forward mutagenesis reporters (typically lacZ or luciferase-based). Aliquots of these cultures can be sub-cultured in the presence of a selection to carry out directed evolution in real time. 
     All disclosed subject matter is claimed and is not limited by the following embodiments, which are exemplary and not limiting. 
     The invention encompasses a method for providing random mutagenesis by expressing an error-prone allele of Pol I (EP-Pol I, bearing three mutations: I709N, A759R, and D424A engineered into a polA12 strain (Pol A is strain) of  E. coli , and placing a gene of interest in a ColE1 plasmid, when the ColE1 plasmid is replicated by EP I, random mutations are introduced. 
     The invention further encompasses a method for providing random mutagenesis comprising the steps of: 
     (i) Transformation a mutagenic plasmid containing K54E-LF Pol I into the electro competent JS200 (SC18 polA12 recA718 uvr355) cells (temperature-sensitive for Pol I function), 
     (ii) Recovering cells at 30° C. and plating them out on selective media and incubate overnight at 37° C. 
     (iii). Pick a colony and make bench top competent cells by washing them twice in 10% glycerol in a 1.5 Eppendorf tube, 
     (iv) Transforming the ColE1 reporter plasmid containing GFP and the gene of interest into JS200_K54E cells and plating onto plates containing selection antibiotics, 
     (v) Incubating overnight at 37° C. (at this temperature endogenous Pol I is inactive so the primary replication activity comes from K54E-LF Pol I.), 
     (vi) Washing the plate, and using the plate wash to inoculate liquid selective media, and repeated passage of culture into fresh media, 
     (vii) Prepare minipreps (or similar) of each of the saturated cultures, 
     (viii) Take minipreps and transform into TOP 10 (or similar) cells, 
     (ix) Plate at different concentrations so as to get a ˜500 colonies per plate. 
     (x) Observe under UV light and grade “mutation index” reflecting the diversity of fluorescent signal wherein the number of colonies whose fluorescence is visibly inferior to that of the average “dark colonies” and the number of colonies whose fluorescence is visibly superior to that of the average colony “superbright colonies” is scored according to the following relative scale:
         0=no evidence of mutagenesis   1=rare dark colonies   2=some dark and superbright colonies   3=about 10% dark colonies, frequent superbright   4=10-30% darks many superb rights   5=˜50% darks many superbright   6=majority darks   7=almost all darks       

     Further, the invention encompasses DNA polymerase I having a missense mutation at position 54 (K54E) of its 5′ to 3′ exonuclease domain that enhance continuous mutagenesis. A Pol A polymerase carrying similar acidic residues at positions lining up with  E. coli  K54 
     Additionally, the invention encompasses a DNA polymerase having the following point mutations: K54E, I709N, A759R, and D424A or combinations thereof, or similar amino acid substitutions at homologous position Pol A polymerases. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows six conserved regions were identified through the multiple sequence alignment of the bacterial and bacteriophage nucleases. 
         FIG. 2  is a map of the plasmid used, indicating the location of GFP, kanamycin and carbenicillin resistance. 
         FIG. 3  shows average mutation index scores for increasing passage, with white representing day 2, light grey day 4, grey day 6 and black day 10. While in this figure K54 doesn&#39;t seem to be performing substantially better than the parental EP1, when we looked at individual clones, it did. When we looked at individual clones, it is also clear that only a subset shows continuous mutagenesis, about half of them don&#39;t, consistent with our previous observations of lack of continuous mutagenesis. 
         FIG. 4  shows graphs for EP1 and K54E clones mutation index scores against time. 
         FIG. 5  shows pictures of the actual plates for K54E clone No. 3 under UV showing a consistent increase in the number of dark and dim colonies with increasing passage in culture. 
     
    
    
     DETAILED DESCRIPTION 
     The following publications may be of use understanding the background to and supporting the present invention and are all incorporated by reference for all purposes: Camps et al: PNAS Aug. 19, 2003, vol. 100 no. 17, 9727-9732; Alexander et al. (incl. Camps): Methods Mol. Biol. 2014; 1179: 31-44. doi:10.1007/978-1-4939-1053-3; Labrou. Current Protein and Peptide Science, 2010, 11, 91-100. 
     An exemplary embodiment of the method of the invention is as follows. Note that when specific cell lines, temperatures, volumes, times and apparatus (etc.) are mentioned, any reasonable variation may be used. 
     Transformation of Error-Prone Polymerase 
     1. Transform mutagenic plasmid containing K54E-LF Pol I (novel mutant) into the electro competent JS200 (SC18 polA12 recA718 uvr355) cells. Note that any other suitable competent cell line may be used as will be within the understanding of one skilled in the art. These are temperature-sensitive for Pol I function. 
     2. Recover cells at 30° C. (Note that any other suitable temperature may be used such as from 12-40° C., 15-30° C., 20-15° C. etc.) and plate on nutrient media plates, e.g., LB containing appropriate selective media (examples include antibiotics kanamycin, carbenicillin or zeocin, tetracycline etc. functional complementation, or addiction system) and incubate overnight at 37° C. (or there abouts). In certain embodiments the temperature may vary from between 35° C. and 42° C. 
     3. Pick a colony and make bench top competent cells by washing them twice in 10% glycerol in a 1.5 eppendorf tube (or similar method). 
     Mutagenesis: 
     4. Transform the ColE1 reporter plasmid containing GFP and the gene of interest into JS200_K54E cells and plate onto pre-warmed nutrient media plates, e.g., LB plates (37° C.) containing selection antibiotics. Note that any other suitable reporter plasmid with a suitable fluorescent, immunological, radiological etc. reporter may be used as will be within the understanding of one skilled in the art. 
       FIG. 2  is a map of the plasmid used, indicating the location of GFP, kanamycin and carbenicillin resistance. This is an example representative of any Pol I-dependent plasmid with a selectable marker and a mutagenesis reporter. The mutagenesis reporter is in principle optional, but allows monitoring the experiment. 
     5. Incubate the Petri dish(es) overnight (or for various times between about 8 hrs. and 60 hrs.) at 37° C. (at this temperature endogenous Pol I is inactive so the primary replication activity comes from K54E-LF Pol I.) In various embodiments the temperature range may be between 35° C. and 42° C. 
     6. Wash plate with about 2 mL of media such as LB broth and take a volume, for example 5 ul of plate wash to inoculate some amount, for example 5 ml of selective media, and keep passing some amount, for example 5 μl (microliters) of saturated culture into fresh media. 
     7. Take the rest of the plate wash and miniprep (or similar procedure) and also miniprep (or similar procedure) each of the saturated cultures. 
     8. Readout: Take minipreps and transform into TOP 10 cells or any strain of  E. coli  bearing a WT allele of Pol I (or any other suitable strain) so that plasmids are separated individually and expressed in high copy number. 
     9. Plate at different concentrations so as to get about 500 colonies per plate (informative range is between 100 and 2500 colonies). 
     10. Observe under UV light and grade “mutation index” reflecting the diversity of fluorescent signal according to the following key:
         0=no evidence of mutagenesis 1=rare dark colonies   2=some dark and superlight colonies   3=about 10% dark colonies, frequent superbright   4=10-30% darks many superbrights   5=50% darks many superbright   6=majority darks   7=almost all darks       

     The results are shown in  FIG. 3 , where white columns represent day 2 passage, light grey day 4, grey day 6 and dark grey day 10. Note that the terms above are relative to one another so super-bright is brighter than Blight which is brighter than dark. 
       FIG. 3  shows average mutation index scores for increasing passage, with white representing day 2, light grey day 4, grey day 6 and black day 10. While in this figure K54 doesn&#39;t seem to be performing substantially better than the parental EP1, when we looked at individual clones, it did. When we looked at individual clones, it is also clear that only a subset shows continuous mutagenesis, about half of them don&#39;t, consistent with our previous observations of lack of continuous mutagenesis.  FIG. 4  shows graphs for EP1 and K54E clones mutation index scores against time. 
       FIG. 5  shows pictures of the actual plates for K54E clone No. 3 under UV showing a consistent increase in the number of dark and dim colonies with increasing passage in culture. 
     ADVANTAGES OF THE INVENTION 
     This invention allows performing evolution in real time, i.e. allows performing a selection without having to do a library prep first because mutations are introduced into these cell&#39;s DNA at the same time as they undergo selection. A significant advantage is that there is No need for iteration, which greatly facilitates the exploration of long trajectories in sequence space. Iterative mutagenesis and selection (the way directed evolution is currently done) is labor intensive and each round of mutagenesis inactivates part of the library. Continuous mutagenesis coupled to a strong selection avoids this by ensuring all mutants present have a minimum of activity. The other critical advantage is that this invention is scalable, i.e. the number of mutants that can be explored is only limited by the size of the culture, not by ligation or amplification steps. Commercial advantages include the fact that this method is much cheaper than the current methods because there is no need for cloning, and it is highly scalable. 
     Disadvantages include restricted genetic diversity. The main disadvantage is that, depending of the strength of selection, libraries can experience severe bottlenecks, restricting exploration of sequence space. Clonal interference, where a number of clones with similar moderate increases in fitness compete against each other, also restricts genetic diversity. Finally, only trajectories where each mutation increases fitness are effectively explored. In our systems, fitness “valleys” (i.e. combinations of mutations with a negative impact on fitness) represent barriers for exploration that other systems overcome by increasing the mutation load. Another disadvantage is un-targeted mutagenesis. Mutagenesis is not targeted to the ORF of the gene of interest or to specific areas within that gene, so quantitative effects on activity can be due to regulation of gene expression or plasmid copy number. Qualitative effects (gain of function), however, should be largely insensitive to increased gene expression. So our method is especially adequate for the evolution of new genetic activities rather than for modulation of existing ones. 
     GENERAL INTERPRETATION OF THE DISCLOSURE 
     In this specification, reference is made to particular features of the invention. It is to be understood that the disclosure of the invention in this specification includes all appropriate combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular embodiment or a particular claim, that feature can also be used, to the extent appropriate, in the context of other particular embodiments and claims, and in the invention generally. The embodiments disclosed in this specification are exemplary and do not limit the invention. Other embodiments can be utilized and changes can be made. As used in this specification, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a part” includes a plurality of such parts, and so forth. The term “comprises” and grammatical equivalents thereof are used in this specification to mean that, in addition to the features specifically identified, other features are optionally present. The term “consisting essentially of” and grammatical equivalents thereof is used herein to mean that, in addition to the features specifically identified, other features may be present which do not materially alter the claimed invention. The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example “at least 1” means 1 or more than 1, and “at least 80%” means 80% or more than 80%. The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number) or “(a first number)-(a second number), this means a range whose lower limit is the first number and whose upper limit is the second number. Where reference is made in this specification to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously, and the method can optionally include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps. Where reference is made herein to “first” and “second” features, this is generally done for identification purposes; unless the context requires otherwise, the first and second features can be the same or different, and reference to a first feature does not mean that a second feature is necessarily present (though it may be present). Where reference is made herein to “a” or “an” feature, this includes the possibility that there are two or more such features (except where the context excludes that possibility). 
     The phrases “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single stranded or double stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. 
     “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame. 
     A “variant” of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastp with the “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length. A variant may be described as, for example, an “allelic” (as defined above), “splice,” “species,” or “polymorphic” variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass “single nucleotide polymorphisms” (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state. 
     A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.