CN107418964B

CN107418964B - Phage-assisted multi-bacterium continuous directed evolution system and method

Info

Publication number: CN107418964B
Application number: CN201610349254.3A
Authority: CN
Inventors: 刘陈立; 赖旺生; 魏婷; 赵国屏
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2016-05-24
Filing date: 2016-05-24
Publication date: 2021-04-20
Anticipated expiration: 2036-05-24
Also published as: CN107418964A

Abstract

The invention discloses a phage-assisted multi-bacterium continuous directed evolution system and a method, wherein the system comprises a phage SM carrying a target gene to be evolved, and a plurality of helper plasmids HP and induced mutant plasmids IP which support different SM proliferation before and after evolution. Different HP is placed in different host bacteria, so that the multiplication and evolution efficiency of the phage can be effectively improved, mutual interference among different gene elements is avoided, the method has better practicability, and can be used for directed evolution of various genes.

Description

Phage-assisted multi-bacterium continuous directed evolution system and method

Technical Field

The invention relates to the field of biological evolution, in particular to a phage-assisted multi-bacterium continuous directed evolution system and a phage-assisted multi-bacterium continuous directed evolution method.

Background

Directed evolution (also known as laboratory evolution) is a powerful technical approach that can produce biomolecules with defined functions by harnessing the biological evolution process. The generated biomolecules are widely applied to the fields of industrial production, bioengineering, medicine development and the like. The David R Liu laboratory, harvard university, developed a continuous evolution system based on phage growth (phage assisted continuous evolution, PACE). It mainly comprises three modules of SM, HP and IP: phage module (SM): the gIII gene required by packaging and infecting host bacteria in the bacteriophage M13 gene is cut off and inserted into the gene of the biomolecule to be evolved, wherein after the bacteriophage lacks gIII, the host cannot be infected to generate progeny bacteriophage; mutagenesis module (IP): arabinose is used as an inducer to induce the expression of DNAQ926, dam and seqA, so that wrong bases introduced by polymerase in the DNA replication process cannot be cut off, the mutation rate is improved, and the mutation rate of phage is improved by hundreds of times by IP induced by arabinose; auxiliary module (HP): contains gIII gene required by phage to infect host cell for propagation, and determines whether each mutant SM can generate progeny phage SM with infection activity by binding gIII expression on HP and biological activity of target gene to be evolved on SM (for example, if a certain RNA polymerase is evolved to a specific direction, a promoter consistent with the evolution direction is used to control gIII expression). In the present invention, the ability (or inability) of SM or a mutant strain thereof to promote the expression of the gIII gene means that the gene of interest to be evolved on SM or a mutant strain thereof can (or cannot) promote the expression of the gIII gene.

In the evolution pool, when phage carrying a wild type target gene to be evolved infects host cells, genetic material wild type SM is injected into host bacteria, and a replication system in the host bacteria is utilized to replicate the genetic material. At the same time, DNAQ926, dam and seqA on IP were expressed in the host cell under the induction of arabinose, resulting in the mutation of SM. If the mutation acquired in SM (i.e., the mutation of the gene of interest in SM) can initiate the expression of the gii protein, progeny phages with infectivity can be generated. These newly generated progeny phage mutant strains SMe are secreted out of the host bacteria to infect new host bacteria for the next round of replication and propagation. Thus, while SMe mutants that are capable of promoting gIII expression can proliferate continuously and increase their numbers, wild-type SM and mutant SM that is not capable of promoting gIII expression cannot secrete progeny phage for proliferation and do not increase their numbers. In this case, if new host bacteria culture is continuously added into the evolution pond and old culture is discharged, wild-type SM which can not start the expression of gIII or has low expression capacity and mutant strain thereof are quickly eluted, and the mutant strain of SMe which can efficiently start the expression of gIII is finally retained.

Since only evolved SMe was able to initiate gIII expression and proliferate, the original wild-type SM was not able to be directly eluted. However, since the evolution and screening of SM are carried out during the proliferation of SM, the efficiency of SM evolution is extremely low. One solution to this is to introduce two auxiliary modules HP1 (or HP1.1), HP2 into the host bacteria. The gIII on HP1 can be directly initiated by wild type SM, and the gIII on HP2 can only be initiated by evolved SMe. Thus, wild-type SM can support self proliferation by promoting gii expression on HP1, and carry out mutation evolution during proliferation, thereby evolving SMe capable of promoting gii expression on HP 2. The obtained SMe can be subjected to further evolutionary screening through other ways.

In practical applications, the use of the evolutionary system in which the wild-type SM-recognized promoter HP1 and the evolved SMe-recognized promoter HP2 are both present in a host strain is not ideal. HP1 and HP2 were highly similar in function and expression. When they are present in one host bacterium at the same time, serious mutual interference occurs. It often occurs that the initial addition of wild-type SM has a low effect on the initiation of gIII gene expression in HP1 and HP2, and even gIII gene expression in HP1 and HP2 is difficult to initiate, thus making directed evolution difficult.

Disclosure of Invention

The invention provides a phage-assisted multi-bacterium continuous directed evolution system and method, aiming at solving the problems that the propagation and evolution efficiency of phage in a phage assisted evolution system is low and different gene elements identified by phage SM (or target genes carried by SM) before and after evolution interfere with each other. In the present invention, helper plasmids HP of different species are placed in different host bacteria, and cooperate with each other to support efficient proliferation and evolution of phage carrying target genes.

According to a first aspect of the present invention, there is provided a phage-assisted multi-bacterial continuous directed evolution system comprising a phage SM carrying a gene of interest to be evolved, and a host bacterium, wherein said phage SM is deficient in proliferation.

As a further improvement of the invention, the host bacteria in the above system comprise two or more host bacteria supporting phage proliferation and evolution, and the functions are realized by carrying corresponding genetic elements.

As a further improvement of the present invention, the above host bacterium is a natural host bacterium of a non-defective strain of said bacteriophage SM before carrying the corresponding genetic element or a strain obtained by genetic modification of the natural host bacterium, or a non-natural host bacterium which has been genetically modified to obtain a susceptibility.

As a further improvement of the invention, the host bacteria in the system comprise host bacteria S1 and S2; the above-mentioned host bacterium S1 contains helper plasmid HP1 and induced mutation plasmid IP which support the proliferation of SM before evolution, the above-mentioned host bacterium S2 contains helper plasmid HP2 and induced mutation plasmid IP which support the proliferation of SM after evolution; the function of the gene of interest before evolution is linked to the function of the genetic elements supporting the proliferation of the pre-evolved SM on HP1, and the function of the gene of interest after evolution is linked at least in part to the function of the genetic elements supporting the proliferation of the post-evolved SM on HP 2.

As a further improvement of the invention, the system also comprises a host bacterium S3, the host bacterium S3 comprises a helper plasmid HP2 for supporting the proliferation of the SM after evolution and a mutation-inducing plasmid IP, and the helper plasmid HP3, the functions of the gene elements on the HP3 are related to the functions of the target gene before evolution, and the HP3 has functional defects and cannot support the proliferation of the SM before evolution.

In a preferred embodiment of the present invention, the bacteriophage SM in the above system is a temperate bacteriophage.

As a preferred embodiment of the present invention, one or more genes necessary for the propagation of the above bacteriophage SM are excised; accordingly, the helper plasmid contains the excised gene.

In a preferred embodiment of the present invention, the bacteriophage SM is a filamentous bacteriophage.

As a preferred embodiment of the present invention, the above-mentioned bacteriophage SM is M13 bacteriophage in which the gIII gene required for packaging and infecting a host bacterium is excised; accordingly, the above helper plasmid comprises the above gIII gene.

In a preferred embodiment of the present invention, the host bacterium is Escherichia coli carrying factor F.

As a preferred embodiment of the present invention, the target gene is any one or more of a protein-encoding gene and a non-encoding gene.

In a preferred embodiment of the present invention, the target gene is one or more selected from the group consisting of T7RNA polymerase gene, protease gene, cellulase gene, fluorescent protein gene, and densitometric gene.

In a preferred embodiment of the present invention, the mutation-inducing plasmid IP contains a mutagenic gene capable of increasing the mutation rate of genetic information during the transmission process such as replication and transcription.

In a preferred embodiment of the present invention, the mutation-inducing plasmid IP contains a mutagenic gene selected from at least one of DNAQ gene mutant DNAQ926 gene in which the 12 th and 14 th amino acids are mutated to Ala, deoxyadenylate methylase dam gene, hemimethylated GATC binding protein seqA gene, and activation-induced cytosine deaminase gene AID.

According to a second aspect of the present invention, there is provided a phage-assisted multi-bacterial continuous directed evolution method comprising: propagating bacteriophage SM carrying the target gene to be evolved in a culture system containing host bacteria S1 and S2 in the presence of an inducer until bacteriophage SMe carrying the target gene after evolution is generated; wherein the bacteriophage SM has a proliferation defect, the host bacterium S1 comprises a helper plasmid HP1 supporting the proliferation of the pre-evolution SM and a mutation-inducing plasmid IP, and the host bacterium S2 comprises a helper plasmid HP2 supporting the proliferation of the post-evolution SM and a mutation-inducing plasmid IP; the function of the gene of interest before evolution is linked to the function of the genetic elements supporting the proliferation of the pre-evolved SM on HP1, and the function of the gene of interest after evolution is linked at least in part to the function of the genetic elements supporting the proliferation of the post-evolved SM on HP 2.

As a further improvement of the present invention, the method further comprises: propagating bacteriophage SMe carrying the evolved target gene in a culture system containing host bacteria S3 and S2 in the presence of an inducer until bacteriophage SMeN carrying a further evolved target gene is produced, wherein the host bacteria S3 comprises helper plasmid HP2 and mutation-inducing plasmid IP for supporting the proliferation of the evolved SM, and helper plasmid HP3, the function of the genetic elements on HP3 is linked to the function of the pre-evolved target gene, and HP3 has a functional defect and cannot support the proliferation of the pre-evolved SM.

As a further improvement of the present invention, the method further comprises: new host bacteria cultures are added continuously and old cultures are drained.

As a further improvement of the present invention, the method further comprises: and (3) real-time monitoring is carried out through plaque counting or reporter genes so as to display the activity of the target genes and the propagation activity of the bacteriophage.

In the invention, different types of helper plasmids HP are placed in different host bacteria, and the helper plasmids HP are matched with each other to support the efficient propagation and evolution of the phage carrying the target gene. Meanwhile, the method separates the two genes from each other in space and blocks the possibility of mutual interference of the two genes, so that the evolution efficiency can be effectively improved, and the method has important supplement and promotion effects on the research of directed evolution.

Drawings

FIG. 1 is a schematic diagram of the evolution experiment of the directed evolution of the present invention, namely, the positive screening (a) and the negative screening (b).

FIG. 2 is a genetic map of bacteriophage SM in one embodiment of the present invention.

FIG. 3 is a genetic map of plasmid HP1 in one embodiment of the present invention.

FIG. 4 is a genetic map of plasmid HP1.1 in one embodiment of the present invention.

FIG. 5 is a genetic map of plasmid HP2 in one embodiment of the present invention.

FIG. 6 is a genetic map of plasmid HP3 in one embodiment of the present invention.

FIG. 7 is a genetic map of plasmid IP in one embodiment of the present invention.

FIG. 8 is a comparison of SM proliferation in two different host bacteria with two helper plasmids in the same host bacteria in one embodiment of the invention.

FIG. 9 is a comparison of the plaque formation of the evolved product SMe under selection in the host strain S5 carrying the T7 promoter and the host strain S6 carrying the T3 promoter, respectively, in one example of the present invention.

FIG. 10 shows the ratio (S6/S5) of plaques formed by the negative selection of the evolved product in the host strain S6 and the host strain S5, wherein the host strain S6 carries gIII under the control of T3 promoter and the host strain S5 carries gIII under the control of T7 promoter.

FIG. 11 shows the ratio (S7/S6) of plaques formed by the negative selection of the evolved product in the host strain S7 and the host strain S6, wherein the host strain S7 carries gIII under the control of the T3 promoter and gIII-R5 under the control of the T7 promoter, and the host strain S6 carries gIII under the control of the T3 promoter.

FIG. 12 is an alignment of the amino acid sequences of the genes of interest carried by the phage in different products of evolution according to one embodiment of the invention.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.

As shown in FIG. 1, the phage-assisted multi-bacterial continuous directed evolution system according to one embodiment of the present invention includes two parts, positive selection (a) and negative selection (b). It should be noted that, from the implementation of the present invention, only the part (a) of the forward screening is required to implement the goal of transforming the target gene to be evolved into the evolved target gene. Therefore, the negative screening (b) can be regarded as a further improved technical solution of the directed evolution system of the present invention. In fact, the positive screen (a) achieves qualitative changes from the "target gene to be evolved" to the "target gene after evolution", while the negative screen (b) achieves quantitative changes that further enhance the function of the "target gene after evolution".

In the present invention, the "gene of interest to be evolved" may be equivalent to the "gene of interest before evolution", that is, the gene of interest in which a mutagenic mutation has not occurred by the system and method of the present invention, and may be referred to as a "wild-type gene" in some cases. The "evolved target gene" is a gene which has a new function evolved as compared with the "target gene to be evolved", but the "evolved target gene" may have the original function. Of course, the original function may be completely lost. In short, in the present invention, a target gene is referred to as an "evolved target gene" as long as the target gene has a new function that has evolved, regardless of whether the original function is present.

It should be noted that the symbols in the present invention, such as SM, HP, IP, S, etc., are only exemplary symbolic representations. The objects represented by these symbols can be fully translated into other expressions. For example, bacteriophage SM, may be referred to as a pre-evolutionary bacteriophage; phage SMe, which can be referred to as evolved phage; the bacteriophage SMeN, which may be referred to as a phage after further evolution, where the number N (e.g. 1, 2, 3 or 4, etc.) represents the number of generations of subculture; host bacterium S1, which may be referred to as a first host bacterium; host bacterium S2, which may be referred to as a second host bacterium; host bacterium S3, which may be referred to as a third host bacterium; helper plasmid HP1, which may be referred to as the first helper plasmid; helper plasmid HP2, which may be referred to as the second helper plasmid; helper plasmid HP3, which may be referred to as the third helper plasmid; the mutation-inducing plasmid IP may be referred to as a mutation-inducing plasmid.

In addition, the host bacteria of the present invention may include other host bacteria in addition to the above host bacteria S1, S2 and further host bacteria S3. In short, in the present invention, at least the helper plasmid HP1 and the helper plasmid HP2 need to be placed in two host bacteria, respectively. In the present invention, the host bacteria S1, S2, and S3 do not mean "species" but mean helper plasmids carried by the strains. In the present invention, the host bacteria S1, S2 and S3 may be obtained by introducing different helper plasmids into the same species, such as E.coli carrying factor F.

As shown in FIG. 1, the phage-assisted multi-bacterial continuous directed evolution system according to one embodiment of the present invention comprises a phage SM carrying a gene of interest to be evolved, wherein the phage SM has a proliferation defect, by which is generally meant a functional defect in packaging and/or infecting host bacteria, possibly due to mutation of the relevant gene, which differs among different phages with respect to the gene responsible for packaging and/or infecting the host bacteria. For example, in one embodiment of the invention, bacteriophage SM is the M13 bacteriophage in which the gIII gene required for packaging and infection of the host bacterium is excised, resulting in failure to package and infect the host bacterium. It will be appreciated by those skilled in the art that any similar bacteriophage can be used as the bacteriophage SM in the present invention, and is not limited to the M13 bacteriophage.

The directed evolution system shown in FIG. 1 also includes host bacteria S1 and S2. Wherein, the host bacterium S1 contains a helper plasmid HP1 supporting the proliferation of the pre-evolution SM and a mutation-inducing plasmid IP, and the host bacterium S2 contains a helper plasmid HP2 supporting the proliferation of the post-evolution SM and a mutation-inducing plasmid IP. The support of the pre-evolution SM proliferation by the helper plasmid HP1 is achieved by the functional association of the target gene before evolution with the function of the genetic element supporting the pre-evolution SM proliferation on HP 1; similarly, the helper plasmid HP2 supports post-evolution SM proliferation by functionally associating at least part of the evolved gene of interest with the functional elements of the gene supporting post-evolution SM proliferation on HP 2. By "at least in part" is meant that the function of the evolved gene of interest may be related to the function of the pre-evolved SM propagated gene element in addition to a portion of the function related to the function of the post-evolved SM propagated gene element. For example, in one embodiment of the present invention, the gene of interest before evolution refers to T7RNA polymerase gene, the gene of interest after evolution refers to a gene having T3RNA polymerase function (also including T7RNA polymerase function), the gene element on HP1 refers to T7 promoter, and the gene element on HP2 refers to T3 promoter, which respectively control downstream gii gene expression to aid in propagation of the gii functionally deficient phage SM. Thus, the T7RNA polymerase gene was functionally linked to the T7 promoter, while the T3RNA polymerase gene was functionally linked to the T3 promoter.

It will be appreciated by those skilled in the art that the present invention is not limited to the above-described example in which "T7 RNA polymerase gene" is used as the target gene before evolution, but encompasses any similar technical solutions. Specifically, for different target genes, such as protease gene, cellulase gene, fluorescent protein gene and density sensing gene, their functions can be related to the functions of the helper plasmid by different principles.

For ease of understanding, the present invention briefly describes methods for correlating the activities of several other genes of interest with gIII on HP. It should be noted that there are many methods for correlating the activity of the target gene with gIII, and the methods are not limited to these methods. For protease genes: (1) the gIII gene is expressed by fusion with a helper protein (e.g., G6 protein of M13 phage) that blocks binding to the gIII protein, using the degradation sequence of the protease of interest as a linker. The gIII portion of the fusion protein is now blocked and inactive. Only when the protease of interest develops a specific activity, degrading the designated degradation sequence, will active gIII be released. (2) The target protease degradation sequence is used as a connecting fragment by using T7 polymerase as an intermediary, and the T7 polymerase and the T7 lysozyme are subjected to fusion expression to form a new polymerase. This new polymerase activity was blocked by T7 lysozyme. Only when the evolved protease recognizes the designed degradation sequence and cleaves the lysozyme moiety, the resulting polymerase is active. For the cellulase gene: the lactose operon, which is inhibited by the enzymatic hydrolysis product of cellulose, glucose, can be used to control the expression of the gIII gene. For the fluorescent protein gene: photoinduced promoters sensitive to the fluorescence emitted by the fluorescent protein of interest can be used to initiate gIII gene expression. For the density-sensitive gene: a density sensing system can be used to control the gii gene expression.

The negative screen (b) section in fig. 1 shows a further improved solution of the present invention. The system also included a host bacterium S3, which host bacterium S3 contained the helper plasmid HP2 and mutation-inducing plasmid IP that supported proliferation of post-evolution SM, and the helper plasmid HP3, wherein the helper plasmid HP2 and mutation-inducing plasmid IP are the same as in part (a) of the positive screen; however, the function of the genetic element on HP3 correlates with the function of the gene of interest before evolution, while HP3 has a functional defect and cannot support the proliferation of SM before evolution. In one embodiment of the invention, the genetic element on the helper plasmid HP3 is the T7 promoter, the T7 promoter controls the expression of the gIII-R5 gene, and gIII-R5 is defective gIII, which lacks about 70 amino acids between aa280 and aa350 of the gIII gene and cannot support SM proliferation evolution. It will be appreciated by those skilled in the art that gIII-R5 is also exemplary, and that different genes may be used depending on the phage.

In a preferred embodiment of the present invention, the mutation-inducing plasmid IP comprises a mutagenic gene selected from at least one of DNAQ gene mutant DNAQ926 gene in which amino acids 12 and 14 are mutated to Ala, deoxyadenylate methylase dam gene, hemimethylated GATC binding protein seqA gene, activation-induced cytosine deaminase gene AID, which are capable of interfering with the DNA replication process, resulting in an increased mutation frequency. In fact, any gene that can increase the efficiency of mutation can be used as a mutagenized gene in the present invention.

The directed evolution method provided by the invention comprises the following steps: propagating bacteriophage SM carrying the target gene to be evolved in a culture system containing host bacteria S1 and S2 in the presence of an inducer until bacteriophage SMe carrying the target gene after evolution is generated; wherein the bacteriophage SM has a proliferation defect, the host bacterium S1 comprises a helper plasmid HP1 supporting the proliferation of the pre-evolution SM and a mutation-inducing plasmid IP, and the host bacterium S2 comprises a helper plasmid HP2 supporting the proliferation of the post-evolution SM and a mutation-inducing plasmid IP; the function of the gene of interest before evolution is linked to the function of the genetic elements supporting the proliferation of the pre-evolved SM on HP1, and the function of the gene of interest after evolution is linked at least in part to the function of the genetic elements supporting the proliferation of the post-evolved SM on HP 2.

As a further improvement of the above method, the method further comprises: propagating bacteriophage SMe carrying the evolved target gene in a culture system containing host bacteria S3 and S2 in the presence of an inducer until bacteriophage SMeN carrying a further evolved target gene is produced, wherein the host bacteria S3 comprises helper plasmid HP2 and mutation-inducing plasmid IP for supporting the proliferation of the evolved SM, and helper plasmid HP3, the function of the genetic elements on HP3 is linked to the function of the pre-evolved target gene, and HP3 has a functional defect and cannot support the proliferation of the pre-evolved SM.

In addition, by continuously adding new host bacterium culture and discharging old culture, it is possible to elute phage carrying wild-type target gene and screen mutant phage carrying highly active target gene. Specifically, the culture in the evolution system can be spontaneously or passively renewed by continuously adding a new host bacterium culture and discharging the old culture by a mechanical device or by manually continuously adding a new host bacterium culture and discharging the old culture. In addition, the activity of the target gene and the propagation activity of the phage can be displayed by plaque counting or real-time monitoring of a reporter gene.

The present invention will be described in detail below by taking as an example the case where T7RNA polymerase gene T7RNHP recognizing T7 promoter (SEQ ID NO: 1) is evolved into T3 polymerase gene T3RNHP recognizing T3 promoter (SEQ ID NO: 2), but the protection of the present invention is not limited to the evolution of T7RNA polymerase gene. The T3 polymerase used herein is a polymerase functionally recognized by T3 promoter obtained by evolution test, but does not mean that the polymerase has the same gene sequence as the native T3RNA polymerase. The target gene to be evolved carried on bacteriophage SM (SEQ ID NO: 3, map as shown in FIG. 2) is T7RNA polymerase gene, gIII gene on helper plasmid HP1(SEQ ID NO: 4, map as shown in FIG. 3) is expressed under the control of T7 promoter, gIII gene on helper plasmid HP2(SEQ ID NO: 5, map as shown in FIG. 5) is expressed under the control of T3 promoter, gIII-R5 on helper plasmid HP3(SEQ ID NO: 6, map as shown in FIG. 6) is expressed under the control of T7 promoter, wherein gIII-R5 is defective gIII which deletes about 70 amino acids between aa280 and aa350 of gIII gene and cannot support SM evolution proliferation.

The phages, plasmids and strains of the invention were provided by David R Liu laboratories and their genetic information has been reported in the relevant literature (Nat Chem biol. 2014March; 10(3): 216-. The host bacterium used in the present invention is E.coli S1030 derived from E.coli DH10B, and the genotype is F' proA + B + Delta (lacIZY) zzf: Tn10 (Tet)^R)lacI^Q1PN₂₅-tetR luxCDE/endA1 recA1 galE15 galK16 nupG rpsL(Str^R)ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC)proBA∷pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaCλ^-。

Coli S1030, and any e.coli carrying factor F may be used as the host bacterium in the present invention. The E.coli S1030 and SM, HP1, HP1.1, HP2, HP3 and IP (shown in figures 2-7) can be obtained by conventional molecular cloning methods such as PCR, enzyme digestion connection, gene recombination and the like according to gene maps and sequences. Molecular cloning methods such as gene recombination, PCR and enzymatic ligation are well known in the art and have been determined to be able to obtain corresponding strains, plasmids and phages. The host bacteria, plasmids and phages according to the invention thus have reproducible characteristics and can be obtained by those skilled in the art by conventional methods. Accordingly, it will be appreciated by those skilled in the art that the present invention satisfies the full disclosure without providing a seed deposit.

The present invention refers to a host bacterium carrying HP1 and IP (SEQ ID NO: 7, map shown in FIG. 7) as S1, a host bacterium carrying HP2 and IP as S2, and a host bacterium carrying HP2, HP3 and IP as S3. In the positive screen, initial SM was subjected to continuous directed evolution in a mixture of S1, S2. The initial SM carried the T7RNHP gene and was able to undergo higher levels of proliferation and mutational evolution in S1. As SM-borne T7RNHP continues to evolve towards T3RNHP, the resulting mutant SM strains, which are capable of promoting gIII expression under the control of the T3 promoter, are secreted extracellularly and infect S2 bacteria in the same system. In S2 strain, the obtained mutant SM is further evolved to improve the promoter activity of the SM on gIII controlled by the T3 promoter in HP2 to obtain mutant phage SMe and a target gene evolved into pre-T3RNHP (figure 1a), and the process is called positive screening.

After evolution, pre-T3RNHP on SMe has higher activity on T7 and T3 promoters. In order to improve the specificity of pre-T3RNHP to the T3 promoter, T3RNHP with low recognition and promotion capacity to the T7 promoter needs to be screened out by evolution. This process is called negative screening.

HP3 is needed for negative screening, and when the S3 and S2 mixed bacteria are infected by the SMe after the evolution through the positive screening, the expression of gIII-R5 can be started by the pre-T3RNHP gene of the objective gene after the evolution on the SMe, so that the proliferation of the gene is inhibited; meanwhile, due to mutual interference of HP2 and HP3, the SMe directly evolves poorly in S3. At this time, SMe was able to proliferate and evolve efficiently only in S2, which is a mixed bacterium. This situation continues until SMe has evolved some new mutant strains of bacteriophage. The new phage mutant strains carry target genes which can efficiently promote the expression of the gIII gene controlled by the T3 promoter on HP2 but not or rarely promote the expression of the gIII-R5 gene controlled by the T7 promoter on HP3, and the activity of the target genes is not influenced by the interference of the simultaneous existence of HP2 and HP 3. These new phage mutants now entered the S3 strain for further evolution and finally evolved to give phage SMe4 carrying the highly specific T3RNA polymerase gene T3RNHP (FIG. 1 b).

The feasibility of the present invention is illustrated by the following examples, which are only exemplary and are intended to illustrate the feasibility of the present invention and are not intended to limit the scope of the present invention.

Example 1 preparation of host bacteria for evolution

1) The host strain S1 carrying HP1 and the IP plasmid was cultured in LB medium containing 50. mu.g/ml carbenicillin and 25. mu.g/ml chloramphenicol resistance at 37 ℃ and 220rpm until OD600 became 0.6. The bacterial liquid is placed at 4 ℃ for standby, and the preservation time is not more than 1 day.

2) The host strain S2 carrying HP2 and the IP plasmid was cultured in LB medium containing 50. mu.g/ml carbenicillin and 25. mu.g/ml chloramphenicol resistance at 37 ℃ and 220rpm until OD600 became 0.6. The bacterial liquid is placed at 4 ℃ for standby, and the preservation time is not more than 1 day.

3) Host bacteria S3 carrying HP2, HP3 and IP plasmids were cultured in LB medium containing 50. mu.g/ml carbenicillin, 50. mu.g/ml spectinomycin and 25. mu.g/ml chloramphenicol resistance at 37 ℃ and 220rpm until OD600 became 0.6. The cells were collected by centrifugation at 12000rpm for 1min and resuspended in an equal volume of LB containing 50. mu.g/ml carbenicillin and 25. mu.g/ml chloramphenicol resistance. The bacterial liquid is placed at 4 ℃ for standby, and the preservation time is not more than 1 day.

4) The protein carrying HP1.1(SEQ ID NO: 8, map as in figure 4), HP2 and IP plasmid of host bacteria S4, in the containing 50 u g/ml carbenicillin, 50 u g/ml spectinomycin and 25 u g/ml chloramphenicol resistant LB medium, at 37 degrees C, 220rpm conditions were cultured until OD600 ═ 0.6. The bacterial liquid is placed at 4 ℃ for standby, and the preservation time is not more than 1 day. HP1.1 is a replacement plasmid for HP1. Compared with HP1, HP1.1 and HP2 have better plasmid compatibility due to the difference between resistance and a replication initiator, and can coexist in the same host bacterium.

5) The host strain S5 carrying the HP1 plasmid was cultured in LB medium containing 50. mu.g/ml carbenicillin resistance at 37 ℃ and 220rpm until OD600 became 0.6. The bacterial liquid is placed at 4 ℃ for standby, and the preservation time is not more than 1 day.

6) The host strain S6 carrying the HP2 plasmid was cultured in LB medium containing 50. mu.g/ml carbenicillin resistance at 37 ℃ and 220rpm until OD600 became 0.6. The bacterial liquid is placed at 4 ℃ for standby, and the preservation time is not more than 1 day.

7) The host strain S7 carrying the HP2 and HP3 plasmids was cultured in LB medium containing 50. mu.g/ml carbenicillin and 50. mu.g/ml spectinomycin resistance at 37 ℃ and 220rpm until OD600 became 0.6. The bacterial liquid is placed at 4 ℃ for standby, and the preservation time is not more than 1 day.

In the following examples, the host bacteria were prepared in example 1, and the OD600 was 0.6.

Example 2 method for observing SM plaque in S5 host bacteria

1) A10-cm bacterial culture plate was covered with 10ml of 2% agarose gel and left at room temperature for 20min to coagulate.

2) When the concentration of SM is unknown, it is necessary to multiply SM by 0 and 10^{1，2，3，4，5}Serial gradient dilutions were performed at double ratio.

3) 200. mu.L of 6 groups of S5 host bacteria prepared in example 1 were collected, and 10. mu.L of SM in "2" with different dilution gradients was added to each group, followed by 4ml of LB medium stored at 55 ℃ and containing 0.4% agar and carbenicillin at a final concentration of 50. mu.g/ml. After mixing on a vortex mixer, the samples were spread onto the plates prepared in "1)".

4) Incubated overnight in a 37 ℃ Biochemical incubator.

5) And observing and counting the number of plaques formed by each gradient dilution sample, and calculating the SM concentration.

Example 3 method for observing SM plaque in S6 and S7 host bacteria

In the same manner as in example 2, the host bacteria capable of forming plaques were replaced with the S6 bacteria and the S7 bacteria prepared in example 1, respectively.

Example 4 proliferation of SM in directed evolution systems when HP1 and HP2 were split into two different host bacteria

1) This example analyzes the proliferation of SM in the directed evolution system when the helper plasmid HP1, whose gIII gene is under the control of the T7 promoter, and the helper plasmid HP2, whose gIII gene is under the control of the T3 promoter, are divided into two different host bacteria.

2) The S1 and S2 bacteria prepared in example 1 were inoculated in the following manner: 1 equal volume mixing, adding 240 μ L of the mixed bacterial liquid and 10 μ L into a 96-well bacterial culture plate⁸PFU (plaque forming Unit) SM (10. mu.L) was incubated at 37 ℃ and 220rpm for 1 hour.

3) In another well, the SM culture product of "2)" was diluted 10-fold with the same mixed bacteria, and then cultured in a 250. mu.L system for 1 hour. After 1h, the culture product was subjected to further 5 successive dilution cultures in the same manner.

4)12000rpm, centrifugation for 1min and supernatant collection. The SM concentration in the supernatant was calculated as in example 2.

5) As a result, as shown in FIG. 8, when HP1 and HP2 were separated into different host bacteria, SM proliferated well, and a higher SM concentration was maintained even after continuous gradient dilution.

Example 5 proliferation of SM in directed evolution System when HP1.1 and HP2 are in the same host bacterium

1) This example analyzes the SM proliferation in the directed evolution system when the helper plasmid HP1.1, whose gIII gene is controlled by the T7 promoter, is in the same host bacterium as the helper plasmid HP2, whose gIII gene is controlled by the T3 promoter.

2) To a 96-well bacterial culture plate, 240. mu.L of the S4 bacterial suspension prepared in example 1 and 10. mu.L of the suspension were added⁸PFU SM (10. mu.L) was incubated at 37 ℃ for 1h at 220 rpm.

3) In another well, the SM culture product of "2)" was diluted 10-fold with the same S4 strain, and the culture was continued for 1 hour in a 250. mu.L system. After 1 hour, the culture product was subjected to further 5 cycles of dilution culture in the same manner.

4)12000rpm, centrifugation for 30min and supernatant collection. The SM concentration in the supernatant was calculated as in example 2.

5) As a result, as shown in FIG. 8, when HP1.1 and HP2 are in the same host bacterium, the proliferation of SM is poor, SM is quickly eluted to an extremely low concentration which can not be detected by manual dilution, and subsequent evolution test cannot be carried out.

Example 6 positive screening evolution of SM in a phage-assisted multiple bacteria continuous directed evolution system.

1) The S1 and S2 bacteria prepared in example 1 were inoculated in the following manner: 1 equal volume of the mixture was mixed and 1% of the final L-arabinose inducer was added. Positive selection was performed in 96-well bacterial culture plates, with a 250 μ L profile for each step in the evolution. The concentration of the L-arabinose inducer mother liquor is 10 percent.

2) To a 96-well bacterial culture plate, 240. mu.L of "1)" the mixed bacterial liquid and 10 were added⁸SM (10. mu.L) was incubated at 37 ℃ for 1h at 220 rpm. SM began to mutate under arabinose induction.

3) In another culture well, the SM culture product of "2)" was diluted 10-fold with the same arabinose-containing mixed bacteria as "1)" and then cultured for 1 hour. After 1h, the culture product was subjected to further 5 successive cycles of dilution culture and evolution test in the same manner.

4) The S1 and S2 bacteria prepared in example 1 were inoculated in the following manner: after 4 volumes of mixing, the l-arabinose inducer was added at a final concentration of 1%.

5) The final culture in "3)" was diluted 5-fold with the "4)" broth and cultured for 1 hour. After 1h, the culture product was subjected to further 5 successive cycles of dilution culture and evolution test in the same manner.

6) The final culture supernatant in "5" was collected by centrifugation at 12000rpm for 1 min. mu.L of the supernatant was mixed with 2.5. mu.L of the S2 strain prepared in example 1, and diluted to 250. mu.L with a fresh medium. And continuously culturing the diluted product for 8h, and screening the amplified evolution product.

7) The final evolution product obtained by the method is marked as SMe, and the carried target gene after evolution is marked as pre-T3 RNHP.

8) Plaque-forming unit concentrations were calculated by observing the SMe in the final culture supernatant for plaque formation in S5 and S6, which control gIII expression by T7 and T3 promoters, respectively, as described in examples 2 and 3, respectively.

9) As shown in FIG. 9, wild-type SM could form plaques only in S5 bacteria in which gIII expression was controlled by the T7 promoter. The evolution product SMe can form plaques in S5 and S6 bacteria of which gIII expression is controlled by a T7 promoter and a T3 promoter, the ratio of the number of the plaques is 3:1, and a good evolution effect is shown. If the activity of the target gene after evolution is represented by the plaque forming amount, the activity of the target gene pre-T3RNHP after evolution carried by SMe on the promoters of T7 and T3 is also 3: 1. In the embodiment, the expected activity of the target gene is successfully evolved through a positive screening evolution test of a phage-assisted multi-bacterium continuous directed evolution system.

Example 7 negative selection evolution of SMe in a phage-assisted multiple bacteria continuous directed evolution system.

1) The S2 and S3 bacteria prepared in example 1 were inoculated in the following manner: after 4 volumes of mixing, 1% of the final L-arabinose inducer and 1mM/L of theophylline were added. Negative selection was performed in 96-well bacterial culture plates, at 250. mu.L per step of evolution. The concentration of the L-arabinose inducer mother liquor is 10 percent. The concentration of the theophylline mother liquor is 150 mM/L.

2) To a 96-well bacterial culture plate, 220. mu.L of "1)" inoculum solution and 30. mu.L of 10. mu.L inoculum solution were added⁸SMe from PFU (as counted by plaques formed by SMe in S6 strain). Incubated at 37 ℃ for 1h at 220 rpm. SMe started mutational evolution under arabinose induction.

3) In another culture well, the culture product of "2)" was diluted 5-fold with the same mixed bacteria as "1)" and then cultured for 1 hour. After 1h, the culture product was further subjected to 5 successive cycles of the same dilution culture. This negative sieve product is referred to as SMe 1.

4) And (3) diluting the final culture product of the '3') by 5 times with the mixed bacteria in the '1'), and then continuing culturing for 7h to fully amplify the evolution product.

5) The culture of "4)" was diluted 5-fold with the same mixed bacteria as "1)" and then cultured for 1 hour. After 1h, the culture product was further subjected to 10 consecutive identical dilution cultures.

6) The supernatant in "5" was collected by centrifugation at 12000rpm for 1 min. mu.L of the supernatant was mixed with 33. mu.L of the mixed bacteria in "1)" and diluted to 250. mu.L with fresh medium. The diluted product was cultured for 7 hours. This negative sieve product is referred to as SMe 2.

7) The supernatant from "6" was collected by centrifugation at 12000rpm for 1 min. The supernatant was diluted 10-fold with the S3 strain prepared in example 1, and the culture was continued for 1 hour. After 1h the culture product was subjected to 5 identical dilution incubations. This negative sieve product is referred to as SMe 3.

8) The final culture of "7)" was diluted 10-fold with S3 strain prepared in example 1 and cultured for 2 hours. After 2h the culture product was subjected to 2 identical dilution incubations. This negative sieve product is referred to as SMe 4.

9) The plaque formation of the negative screening products in S5, S6 and S7 bacteria in each step is detected respectively according to examples 2 and 3, and the evolution effect of the negative screening is analyzed.

As shown in FIG. 10, it was found by comparing the plaque formation of the evolved product in S6 and S5 strains, which control the expression of gIII by T3 and T7 promoters, respectively, that as the screening continued, the plaque formation was stronger in S6 strain containing the T3 promoter and weaker in S5 strain containing the T7 promoter. The number of plaques formed by the final evolution product SMe4 in the S6 and S5 bacteria is close to 20: 1. This indicates that the T3RNA polymerase activity of the target gene T3RNHP on SMe4 was enhanced and the T7RNA polymerase activity was reduced compared to 1:3 before evolution. Through evolution experiments, the specificity of the T3RNHP of the target gene is obviously improved.

Comparing the plaque formation of the evolution product in S7 and S6 bacteria. The S7 strain contains gIII-R5 controlled by the T7 promoter and gIII controlled by the T3 promoter, SMe cannot form plaques in S7 strain before negative selection evolution, and the SMe only forms plaques in S6 strain containing only gIII controlled by the T3 promoter. While the evolution continued, the ability of the final SMe4 to form plaques in the S7 and S6 strains was the same. As shown in FIG. 11, the ratio of plaque formation of the negative-sieve evolution product in S7 and S6 bacteria was gradually increased from 0:1 to 1: 1. This shows that the evolution product T3RNHP of the target gene carried by the phage SMe4 obtained by final evolution has high specific T3RNA polymerase activity, and the non-specific T7RNA polymerase activity is small enough not to affect the T3RNA polymerase activity. In the embodiment, the specificity of the activity of the target gene is successfully improved through a negative screening evolution test of a phage-assisted multi-bacterium continuous directed evolution system.

Example 8 alignment of the sequences of the genes of interest carried by SM, SMe and SMe4

And selecting plaques from SM, SMe and SMe4, sequencing the plaques, and comparing the amino acid series differences of the wild type target gene T7RNHP before evolution and the target gene pre-T3RNHP and T3RNHP after evolution carried by the plaques respectively. As shown in FIG. 12, only two amino acid missense mutations are required for the evolution from T7RNHP to pre-T3 RNHP. While the evolution from T7RNHP to T3RNHP requires 7 amino acid missense mutations.

The present invention was explained in the above examples by evolving the T7RNA polymerase gene T7RNHP which recognizes the T7 promoter into the T3RNA polymerase gene T3RNHP which recognizes the T3 promoter. Other target genes (such as protease genes, cellulase genes, fluorescent protein genes, density induction genes and the like) are used for replacing T7RNHP genes in SM, expression regulation and modification modes after expression of gIII (gIII-R5) genes in HP1, HP2 and HP3 plasmids are correspondingly adjusted, so that the expression of the gIII (gIII-R5) is bound with the biological activity of a new target gene to be evolved on SM, and the system can be used for directed evolution of the new target gene.

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A phage-assisted multi-bacterium continuous directed evolution system, comprising a phage SM carrying a gene of interest to be evolved, and a host bacterium, wherein the phage SM has a proliferation defect;

the host bacteria in the system comprise more than two host bacteria supporting phage multiplication and evolution, the functions of the host bacteria are realized by carrying corresponding gene elements, and the host bacteria are escherichia coli carrying F factors;

before carrying corresponding gene elements, the host bacteria are natural host bacteria of the non-defective strains of the bacteriophage SM or strains obtained after genetic modification of the natural host bacteria, or non-natural host bacteria which obtain susceptibility after genetic modification;

the host bacteria in the system comprise host bacteria S1 and host bacteria S2; the host bacterium S1 comprises a helper plasmid HP1 supporting pre-evolution SM proliferation and a mutation-inducing plasmid IP, and the host bacterium S2 comprises a helper plasmid HP2 supporting post-evolution SM proliferation and a mutation-inducing plasmid IP; the function of the gene of interest before evolution is linked to the function of the genetic elements supporting the proliferation of the pre-evolution SM on HP1, and the function of the gene of interest after evolution is linked, at least in part, to the function of the genetic elements supporting the proliferation of the post-evolution SM on HP 2;

the phage SM is M13 phage, wherein the gIII gene required for packaging and infecting host bacteria is excised; accordingly, the helper plasmid comprises the gIII gene;

the mutation-inducing plasmid IP comprises a mutagenic gene, wherein the mutagenic gene consists of a DNAQ gene mutant DNAQ926 gene with the 12 th and 14 th amino acids mutated into Ala, a deoxyadenylate methylase dam gene and a semi-methylated GATC binding protein seqA gene, and can improve the mutation rate of genetic information in the processes of replication, transcription and other transmission processes.

2. The system of claim 1, further comprising a host bacterium S3, the host bacterium S3 comprising a helper plasmid HP2 and a mutation-inducing plasmid IP that support post-evolution SM proliferation, and a helper plasmid HP3, the function of the genetic elements on HP3 being linked to the function of the gene of interest before evolution, while HP3 has a functional defect and cannot support pre-evolution SM proliferation.

3. The system of claim 1, wherein the target gene is any one or more of a protein-encoding gene and a non-encoding gene.

4. The system of claim 3, wherein the target gene is selected from one or more of T7RNA polymerase gene, protease gene, cellulase gene, fluorescent protein gene and density sensing gene.

5. A phage-assisted multi-bacterial continuous directed evolution method, comprising: propagating bacteriophage SM carrying the target gene to be evolved in a culture system containing host bacteria S1 and S2 in the presence of an inducer until bacteriophage SMe carrying the target gene after evolution is generated; wherein the bacteriophage SM has a proliferation defect, the host bacterium S1 comprises a helper plasmid HP1 and a mutation-inducing plasmid IP that support proliferation of pre-evolution SM, and the host bacterium S2 comprises a helper plasmid HP2 and a mutation-inducing plasmid IP that support proliferation of post-evolution SM; the function of the gene of interest before evolution is linked to the function of the genetic elements supporting the proliferation of the pre-evolution SM on HP1, and the function of the gene of interest after evolution is linked, at least in part, to the function of the genetic elements supporting the proliferation of the post-evolution SM on HP 2;

the host bacterium is escherichia coli carrying F factors;

the method further comprises the following steps: new host bacteria cultures are added continuously and old cultures are drained.

6. The method of claim 5, further comprising: propagating bacteriophage SMe carrying the evolved gene of interest in a culture system containing host bacteria S3 and S2 in the presence of an inducer until bacteriophage SMeN carrying a further evolved gene of interest is produced, wherein said host bacteria S3 comprises helper plasmid HP2 and mutation-inducing plasmid IP supporting proliferation of the evolved SM, and helper plasmid HP3, the function of the genetic elements on HP3 being linked to the function of the pre-evolved gene of interest, while HP3 has a functional defect not supporting proliferation of the pre-evolved SM.

7. The method of claim 5 or 6, further comprising: and (3) real-time monitoring is carried out through plaque counting or reporter genes so as to display the activity of the target genes and the propagation activity of the bacteriophage.

8. The method according to claim 5 or 6, wherein the target gene is any one or more of a protein-encoding gene and a non-encoding gene.

9. The method according to claim 8, wherein the target gene is selected from one or more of T7RNA polymerase gene, protease gene, cellulase gene, fluorescent protein gene and density sensing gene.