Multi-host in-vivo continuous evolution system and method and application thereof

Abstract

The invention belongs to the technical field of synthetic biology, and particularly relates to a multi-host in-vivo continuous evolution system and method and application thereof. Specifically, a target plasmid containing a target gene and an OriV replicon is targeted through combined expression of an initiation protein RepC in a broad-host plasmid RSF1010 replication system, TadA8e-RepA fused with deaminase TadA8e and helicase RepA, and RepB-PolI auxiliary plasmids fused with an initiation enzyme RepB of error-prone PolI, and the invention provides an effective in-vivo continuous evolution method. The method not only can be evolved in escherichia coli cells, but also can be used for other bacteria and fungi, so that the method has considerable application value and prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and particularly relates to a multi-host in vivo continuous evolution system, method and its application. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Directed evolution technology is a very important molecular biology technology. Directed evolution has generated rich biodiversity. Through artificial mutation and screening of target genes, a large number of proteins with improved performance have been produced. Due to the good sustainability of in vivo evolution, it has received a great deal of attention in recent years.

[0004] Currently, all in vivo evolution methods can only be achieved in Escherichia coli or Saccharomyces cerevisiae. However, with the development of synthetic biology, expressing correctly folded and active heterologous proteins / enzymes in any target microbial host is crucial for establishing metabolic functions.

[0005] A perfect in vivo evolution strategy should meet the following requirements: (1) Mutations can be targeted to the target protein; (2) Show a low off-target mutagenesis effect to maintain cell viability; (3) The mutation range covers at least one gene; (4) The mutation frequency is high enough and can cover all types of mutations; (5) Applicable to different hosts. Loeb et al. developed a method to use error-prone PolI to mutate genes on the ColE1 plasmid to achieve targeted mutagenesis and in vivo continuous evolution of genes. However, the use of the complete PolI significantly increases the frequency of global mutations, and this method is only applicable to Escherichia coli. In order for the protein to function efficiently in its host, it is very necessary to perform directed evolution in the corresponding host. Phage-assisted continuous evolution (PACE) achieved more efficient in vivo mutagenesis evolution, but it is still limited by phage culture technology and only applicable to Escherichia coli and other problems, so it has not been widely applied. The in vivo evolution method based on orthogonal replication plasmids and retrotransposons achieved orthogonal mutagenesis of target genes, but its host is limited to yeast. Subsequently, the CRISPR-based EvolvR has the problem of a small mutation range, and the in vivo evolution method based on T7 RNA polymerase and deaminase has the problem of limited mutation types. Currently, there is still no in vivo evolution method that meets all the above requirements. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a multi-host in vivo continuous evolution system, method and its application. Based on a broad-host plasmid that can replicate and combinatorially express in a variety of bacteria and fungi, the present invention establishes a method for multi-host in vivo continuous evolution, which is named "ITMU (An in vivo target mutagenesis system for multiple hosts)". By demonstrating the mutation frequency of the deaminase-helicase and polymerase-primase complex proteins, the mutation rate of ITMU reaches 10 -5 to the level, and the off-target level is relatively low. This method can not only evolve proteins in Escherichia coli hosts, but also be used in other bacteria and fungi, such as Pseudomonas putida, Corynebacterium glutamicum, and Yarrowia lipolytica, with good universality. Based on the above research results, the present invention is completed.

[0007] To achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0008] In the first aspect of the present invention, a multi-host in vivo continuous evolution system is provided. The system includes plasmids, at least two of which, at least including an auxiliary plasmid and a target plasmid;

[0009] The auxiliary plasmid contains the coding gene of the fusion protein TadA8e-RepA of deaminase and helicase, the coding gene of the initiator protein RepC, and the coding gene of the fusion protein RepB-PolI4MT of primase and error-prone PolI;

[0010] The target plasmid contains the coding gene of the target gene and the replicon OriV;

[0011] The deaminase is TadA8e derived from Escherichia coli; the error-prone PolI is a variant of DNA polymerase I derived from Escherichia coli, which contains mutations of D424A, I709N, R751K and A759R on the basis of wild-type DNA polymerase I and contains thioredoxin, named PolI4MT (its amino acid sequence is shown in SEQ ID NO.1). The fidelity of this enzyme is very low and is suitable for generating mutations for directed evolution. The thioredoxin is the thioredoxin from T7 DNA polymerase, located at positions 571-646 in the PolI4MT protein.

[0012] In the second aspect of the present invention, a method for a multi-host in vivo continuous evolution system is provided. The method includes transforming the above plasmids into host cells to achieve continuous evolution of the target protein in the host cell body;

[0013] More specifically, the method includes: using the initiator protein RepC in the broad-host plasmid replication system to bind to the replicon OriV of the plasmid to promote double-strand separation; using the DNA helicase RepA to unwind the double-stranded DNA in two directions, and the deaminase to deaminate the single-stranded DNA; using the primase RepB fused with error-prone PolI to recognize two initiation signals inside OriV and synthesize primers; after the primers are synthesized, RepB separates from the replicon, binds to the primers, starts replicating the plasmid, and introduces mutations into the plasmid, thereby realizing the continuous evolution of the target protein in the host cell body.

[0014] In a third aspect of the present invention, there is provided an application of the above system and / or method in directed evolution of proteins.

[0015] Beneficial technical effects of the above one or more technical solutions:

[0016] The above technical solution discloses an orthogonal multi-host in vivo continuous evolution system, wherein the key enzyme for in vivo continuous evolution is the fusion protein TadA8e-RepA of helicase and deaminase, the initiator protein RepC, and the fusion protein RepB-PolI4MT of primase and error-prone DNA polymerase 1. Compared with other continuous evolution methods, the above technical solution has the characteristics of high mutation frequency, complete mutation types, and simple operation. The present invention only needs to transform the auxiliary plasmid containing the fusion protein TadA8e-RepA of helicase and deaminase, the initiator protein RepC, and the fusion protein RepB-PolI4MT of primase and error-prone DNA polymerase 1, and the target plasmid containing the target gene and the replicon OriV, and can realize the in vivo continuous evolution of the target protein. The present invention can achieve in vivo continuous evolution in a variety of hosts. Specifically, it is applicable to Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, Gram-positive bacteria such as Corynebacterium glutamicum, and eukaryotic microorganisms such as Yarrowia lipolytica and other microorganisms, and can directly carry out in vivo continuous evolution in these microorganisms, improving the frequency of in vivo continuous evolution; therefore, the engineered bacteria have considerable application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0018] Figure 1 This is the design of the multi-host in vivo continuous evolution of the present invention. By cloning the TadA8e-RepA, RepB-PolI4MT, and RepC replication protein genes, an auxiliary plasmid was constructed. The target gene was inserted into the plasmid containing the OriV replicon to form the target plasmid.

[0019] Figure 2 This is the schematic diagram of the multi-host in vivo continuous evolution system of the present invention. Specifically, RepA unwinds the DNA double strand, TadA8e deaminates the bases on the single-stranded DNA, and RepB binds to the DNA and synthesizes primers. After RepB separates from the DNA, the error-prone polymerase I (PolI) binds to the primers, starts replicating the plasmid, and introduces mutations into the plasmid. Since the replication is bidirectional, mutations are introduced into the plasmid in both directions.

[0020] Figure 3 This is the characterization of the mutation rate of the continuous evolution system of the present invention. To obtain the error bars, ten parallel experiments were conducted.

[0021] Figure 4 This is the mutation frequency at different positions of the plasmid for high-throughput sequencing purposes of the present invention.

[0022] Figure 5 This is the proportion of mutation types of the continuous evolution system of the present invention.

[0023] Figure 6 This is the resistance of the evolved strain of Pseudomonas putida L6 to gentamicin of the present invention.

[0024] Figure 7 This is the schematic structural diagram of the mutated sites of Pseudomonas putida L6 by molecular docking simulation of the present invention.

[0025] Figure 8 This is the yield of lycopene produced by the evolved strain of Corynebacterium glutamicum of the present invention. All experiments were repeated four times to obtain error bars.

[0026] Figure 9 This is the growth curve of the xylose-evolved strain of Yarrowia lipolytica of the present invention. All experiments were repeated three times to obtain error bars.

[0027] Figure 10 This is the xylose consumption of the xylose-evolved strain of Yarrowia lipolytica of the present invention. All experiments were repeated three times to obtain error bars.

[0028] Figure 11 This is the schematic structural diagram of the mutated sites of xylose isomerase by molecular docking simulation of the present invention. Detailed implementation manners

[0029] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention.

[0031] As mentioned above, although there are various in vivo continuous evolution methods, these methods all have disadvantages such as insufficient orthogonality, cumbersome operation, host restriction, and single mutation type.

[0032] In view of this, the present invention provides an orthogonal multi-host in vivo continuous evolution system, in which the key enzyme for in vivo continuous evolution is the fusion protein TadA8e-RepA of helicase and deaminase, the starting protein RepC, and the fusion protein RepB-PolI4MT of primase and error-prone DNA polymerase 1. In addition, inserting thioredoxin TBD into DNA polymerase 1 can further increase the mutation frequency of in vivo continuous evolution. Compared with other continuous evolution methods, the above technical solution has the characteristics of high frequency, complete mutation types, and simple operation. The present invention only needs to transform the auxiliary plasmid containing the fusion protein TadA8e-RepA of helicase and deaminase, the coding gene of the starting protein RepC, and the fusion protein RepB-PolI4MT of primase and error-prone PolI, and the target plasmid containing the target gene and the replicon OriV, and can achieve in vivo continuous evolution of the target protein. The present invention can achieve in vivo continuous evolution in multiple hosts.

[0033] Specifically, in a typical specific embodiment of the present invention, a multi-host in vivo continuous evolution system is provided, and the system includes plasmids, and at least two plasmids are included, at least including an auxiliary plasmid and a target plasmid;

[0034] The auxiliary plasmid contains the coding gene of the fusion protein TadA8e-RepA of helicase and deaminase, the coding gene of the starting protein RepC, and the coding gene of the fusion protein RepB-PolI4MT of primase and error-prone PolI;

[0035] The target plasmid contains the coding gene of the target gene and the replicon OriV;

[0036] The deaminase is TadA8e derived from Escherichia coli; the error-prone PolI is a variant of DNA polymerase I derived from Escherichia coli. The variant contains mutations of D424A, I709N, R751K, and A759R on the basis of wild-type DNA polymerase I and simultaneously contains thioredoxin, named PolI4MT (its amino acid sequence is shown in SEQ ID NO.1). The fidelity of this enzyme is very low and is suitable for generating mutations for directed evolution. The thioredoxin is the thioredoxin from T7 DNA polymerase and is located at positions 571-646 in the PolI4MT protein.

[0037] Among them, the target gene can be the coding gene and / or non-coding gene of one or more proteins.

[0038] In a specific embodiment of the present invention, the target gene includes, but is not limited to, resistance genes of antibiotics (such as gentamicin), coding genes of enzymes in catabolic pathways, coding genes of enzymes in anabolic pathways (such as geranylgeranyl pyrophosphate synthase, phytoene synthase, and phytoene desaturase), coding genes of DNA-binding proteins, coding genes of nucleases, coding genes of glycosidases (such as xylose isomerase), and coding genes of proteases. The target gene is applied to continuous evolution.

[0039] In a specific embodiment of the present invention, except for the target gene, the coding genes of deaminase TadA8e and error-prone PolI, the coding genes of the above other enzymes or proteins are all derived from broad-host plasmids. The broad-host plasmids include, but are not limited to, plasmids of IncP, IncN, IncQ, and IncW groups. In a specific embodiment of the present invention, the broad-host plasmid can be the IncQ group plasmid RSF1010.

[0040] In another specific embodiment of the present invention, the distance of the OriV replicon is -4.5 kb - 4.5 kb, thus proving that the above in vivo continuous evolution method is very suitable for the continuous evolution of proteins.

[0041] In another specific embodiment of the present invention, a method for a multi-host in vivo continuous evolution system is provided. The method includes transforming the above plasmid into a host cell to achieve the continuous evolution of the target protein in the host cell body;

[0042] More specifically, the method includes: using the initiator protein RepC in the broad-host plasmid replication system to bind to the replicon OriV of the plasmid to promote double-strand separation; using the DNA helicase RepA to unwind the double-stranded DNA in both directions, and using the deaminase TadA8e to deaminate the generated single-stranded DNA; using the primase RepB fused with error-prone PolI to recognize two initiation signals inside OriV and synthesize primers; after the primers are synthesized, RepB separates from the replicon, and PolI binds to the primers to start replicating the plasmid and introduce mutations into the plasmid, thereby realizing the continuous evolution of the target protein in the host cell in vivo.

[0043] Among them, the broad-host plasmid includes but is not limited to plasmids of the IncP, IncN, IncQ, and IncW groups. In a specific embodiment of the present invention, the broad-host plasmid can be the IncQ-group plasmid RSF1010.

[0044] The deaminase is TadA8e derived from Escherichia coli; the error-prone PolI is a variant of DNA polymerase I derived from Escherichia coli, and the variant contains mutations of D424A, I709N, R751K, and A759R on the basis of wild-type DNA polymerase I and simultaneously includes thioredoxin, named PolI4MT. This enzyme has a very low fidelity and is suitable for creating mutations for directed evolution. The thioredoxin is the thioredoxin from T7 DNA polymerase and is located at positions 571-646 in the PolI4MT protein.

[0045] The host cells include prokaryotic cells and eukaryotic cells;

[0046] More specifically, the prokaryotes include bacteria, and the eukaryotes include fungi.

[0047] The bacteria can be Gram-positive bacteria or Gram-negative bacteria, including but not limited to Escherichia coli, Corynebacterium glutamicum, and Pseudomonas putida.

[0048] The fungi can be Yarrowia lipolytica.

[0049] In another specific embodiment of the present invention, an application of the above system and / or method in protein directed evolution is provided.

[0050] The present invention will be further explained and illustrated by the following examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods not specifically described are all conventional methods. For example, reference can be made to "Molecular Cloning: A Laboratory Manual" (Sambrook and Russell, 2001). Unless otherwise specified, the reagents and materials used in the following examples are all commercially available. The strains used in the examples are Escherichia coli DH5α with a genome sequence number of NZ_CP025520.1; Corynebacterium glutamicum ATCC13032 with a genome sequence number of NC_021352.1; Pseudomonas putida KT2440 with a genome sequence number of NC_002947.4; Yarrowia lipolytica Po1f (ATCC number MYA-2613; genotype MATA ura3-302 leu2-270 xpr2-322 axp2-delta NU49 XPR2::SUC2, purchased from ATCC).

[0051] The Tiangen plasmid miniprep kit and agarose gel DNA recovery kit used in the examples were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the DNA polymerase PrimeSTAR was purchased from TAKARA Co., Ltd.; the plasmids pAcyc184, Pet28a, and PRL1342 were purchased from Addgene. The plasmids pKD3, pkt5-t4, and ylep-Leu were plasmids stored in the laboratory.

[0052] Example 1 Construction and Optimization of In Vivo Continuous Evolution Technology

[0053] (1) Construction of In Vivo Continuous Evolution System

[0054] To construct the helper plasmid, the kanamycin resistance gene from plasmid pET28a, the ColEI replicon from plasmid pACYC184, and RepA, RepB, and RepC from plasmid pRL1342, TadaA8e and error-prone PolI (PolI4MT, containing D424A, I709N, R751K, and A759R mutations and thioredoxin, the amino acid sequence of which is shown in SEQ ID NO.1) were amplified using primers and assembled using Gibson assembly. The constructed plasmid was named pUC-TadA8e-4MT. To construct the target plasmid, plasmid pKD3 was used as the backbone, and the RSF1010 replication protein OriV was amplified from plasmid PRL1342 using primers. The two fragments were ligated together using the Gibson assembly method. To characterize the frequency of reverse mutations in the chloramphenicol resistance gene, the third codon of the chloramphenicol resistance gene was mutated to a stop codon.

[0055] Design and synthesize primers:

[0056] kan-repC-R: 5’-CGAGTTCTTCTGATGTAAGTTAGCGCGAATTGCAAGCTG-3’

[0057] polI-PRL-F: 5’-CGAAAACTGGGATCAGGCGCACTAATTGCGTTGGTACTCACGCCTGTTA-3’

[0058] PolI-R: 5’-TTAGTGCGCCTGATCCCAGTTTTCG-3’

[0059] RepB-PolI-F: 5’-CGGCGGCTCCGGTGGTGGTTCTGGAGGCGGTTCTATGGTTCAGATCCCCCAAAATCCAC-3’

[0060] RepB-F: 5’-CCTTAACCATCTTGACACCCCATTG-3’

[0061] PolI-repB-R: 5’-TCCAGAACCACCACCGGAGCCGCCGCCGCTTCCACCGCCCATGCTGAAATCTGGCCCGC-3’

[0062] repC-kana-F: 5’-GCGCTAACTTACATCAGAAGAACTCGTCAAGAAGGCGA-3’

[0063] repB-colEI-R: 5'-CAATGGGGTGTCAAGATGGTTAAGGCAGCAAAAGGCCAGGAACCGTAA-3'

[0064] CmR-A7T-F: 5'-AGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGTAAAAAATCACTGGATATACCACCGT-3'

[0065] repB-CmR-R: 5'-CAATGGGGTGTCAAGATGGTTAAGGAAAATTACGCCCCGCCCT-3'

[0066] CmR-F: 5'-AAAAAAATTACGCCCCGCCCTG-3'

[0067] CmR-ORIV-R: 5'-TAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTC-3'

[0068] TadA8e-F: 5'-ATGGGTAGCAGCCATCATCATCAT-3'

[0069] TadA8e-R: 5'-TTCTGCAGAACCGCCACCGCCAGAGCCACCACCACCGCTACCACCGCTATTAATGCTGCT-3'

[0070] TBD-F: 5'-TGGCTGAGCTGGAAAAGAAAGCGACCGAAACCTTCGGTTCTTGGT-3'

[0071] TBD-R: 5'-GTAACTGCTTGGTGGAAGAAAGGTTAAAAACAACGTGTTCAACCGGGGTG-3'

[0072] Using plasmids pET28a, pACYC184, pRL1342 as templates, PCR (polymerase chain reaction) amplification was performed with primers PolI-PRL-F / kan-repC-R, RepB-PolI-F / PolI-R, RepB-F / PolI-repB-R, TadA8e-F / TadA8e-F, TBD-F / TBD-R, and repC-kana-F / repB-colEI-R to obtain fragments kana, RepA, RepC, RepB, TadaA8e, TBD, and PolI.

[0073] Using plasmids pKD3 and pRL1342 as templates, PCR (Polymerase Chain Reaction) amplification was performed with primers CmR-A7T-F / repB-CmR-R and CmR-OriV-R / CmR-F to obtain fragments CmR and RSF1010 replication protein OriV.

[0074] PCR reaction conditions: Pre-denaturation at 97°C for 5 min, denaturation at 94°C for 60 s, annealing at 56°C for 30 s, extension at 72°C for 1 min / kb, extension at 72°C for 10 min after 30 cycles, and storage at 4°C.

[0075] After digestion with DpnI endonuclease, recovery and purification were carried out. The above fragment products were ligated using the Gibson Assembly Cloning Kit (New England Biolabs (NEB), England) to construct plasmids pUC-TadA8e-4MT and R6k-OriV-CmR and transformed into Eschrichia coli DH5α strain.

[0076] The electrotransformation method for Eschrichia coli DH5α is as follows:

[0077] 1) Transfer from the preserved E. coli DH5α glycerol tube to a test tube containing 5 mL of LB medium at an inoculation amount of 1%, and culture overnight with shaking at 30°C and 220 rpm in a shaker.

[0078] 2) Transfer at an inoculation amount of 1% to a 300 mL wide-mouth Erlenmeyer flask containing 50 mL of LB medium, and culture with shaking at 30°C and 220 rpm for 2 - 3 hours.

[0079] 3) Cultivate the bacterial solution until the OD600 reaches 0.6 - 0.8. Transfer 1.5 mL of the bacterial solution to a 1.5 mL sterilized centrifuge tube, centrifuge at 12000 rpm for 1 minute, discard the supernatant, and repeat three times.

[0080] 4) Add 1 mL of 10% glycerol to the centrifuge tube, pipette and mix evenly, centrifuge at 12000 rpm for 1 minute, discard the supernatant, and repeat three times.

[0081] 5) Resuspend the cells with 100 μL of electrotransformation buffer, add the fragment or plasmid to be transformed. Transfer the above mixed competent cells to a 2 mm electroporation cuvette, use an electroporator, and perform electroporation at 2400 V, 25 μF, and 200 Ω. The electroporation constant is about 5.0. Add 1 mL of SOC medium, recover and culture at 30°C for 1 hour, spread on a solid LB plate containing 25 μg / mL kanamycin, incubate at 30°C, and after single colonies grow on the plate, perform colony PCR verification with the corresponding primers and then sequencing verification.

[0082] Calculating the Mutation Frequency of the In Vivo Evolution System:

[0083] A helper plasmid containing the chloramphenicol resistance gene CmR* (with a nonsense mutation) and the target plasmid were co-transformed into Escherichia coli. Ten transformants were inoculated into a test tube containing 100 ml of LB medium. After 24 hours, 100 μL of the culture was spread on a plate containing chloramphenicol and kanamycin (to maintain the presence of the helper plasmid). The culture broth was diluted 10 5 or 10 6 times, and 100 μL of the diluted solution was spread on a plate without antibiotics. The mutation frequency of the in vivo evolution system was calculated using the ratio of the restored mutant colonies to the total colonies.

[0084] The number of colonies grown on the chloramphenicol (17 μg / mL) resistance plate was counted using a colony counter. After calculation, the in vivo evolution rate reached 5.5×10 -5 ( Figure 3 ). These results indicate that we can achieve in vivo targeted mutagenesis in Escherichia coli by combining a broad-host-range plasmid with a deaminase and an error-prone DNA polymerase. We named it the multiple-host in vivo continuous evolution system (ITMU).

[0085] (3) Determining the Evolutionary Characteristics of ITMU by High-Throughput Sequencing

[0086] We co-transformed a mutant plasmid co-expressing TadA8e-RepA and RepB-PolI4MT fusion proteins with a target plasmid of 9440 base pairs (bp) into Escherichia coli DH5α. We used next-generation sequencing technology (NGS) to sequence the samples after 5 passages to accumulate mutations, and used the samples at the start of passage as a control. We found that the inducible targeted mutagenesis system (ITMU) increased the mutation frequency in all regions of the target plasmid. By analyzing the regional mutation frequency, we found that the mutation frequency was the highest in the region of 1180 - 2360 base pairs from RSF1010 OriV, indicating that this region might be the mutation hot spot of the target plasmid ( Figure 4 ). ITMU exhibited a broad mutation spectrum, including 12 types of base transitions and transversions, with the mutation from A:T to C:G being the most common, which was due to the high mutation ability of TadA8e ( Figure 5 ). The mutation rate of A:T to C:G caused by PolI4MT was relatively high, probably because these mutations bypassed the mismatch repair system. Overall, next-generation sequencing technology confirmed that ITMU has the advantages of a high mutation rate, covering all types of nucleotide substitutions, and a long mutation frame.

[0087] The high-throughput sequencing method:

[0088] Transform the mutant plasmid and the targeting plasmid into Escherichia coli DH5α. Inoculate the transformed strain into a shake flask containing 50 mL of LB medium with antibiotics and culture it at 37 °C and 220 rpm for 12 hours. Then, take 1 μL of the culture and inoculate it into 1 mL of fresh LB medium and culture it under the same conditions for 12 hours. After repeating the inoculation and culturing 10 times, obtain the precipitate by centrifugation and directly use it as a template for PCR amplification. Divide the targeting plasmid into 16 fragments and amplify them for next-generation sequencing (NGS). To avoid the influence of primers on the mutation rate, adjacent fragments have overlapping regions. Mix the 16 fragments in equimolar amounts. Next-generation sequencing and data processing are completed by the company (Genewiz Suzhou Co., Ltd., China).

[0089] Example 2 Practical application of ITMU in non-model microorganisms

[0090] (1) Evolution of gentamicin in Pseudomonas putida

[0091] Pseudomonas is a common pathogen in clinical practice and is resistant to a variety of antibiotics. The clinical utility of gentamicin as a broad-spectrum antibiotic has prompted efforts to characterize the mutations that confer resistance. Mutations in ribosomal protein L6 are considered the main factors conferring gentamicin resistance. Since the L6 of Pseudomonas does not function in other bacteria, directed evolution of Pseudomonas is necessary to explore the resistance mechanism. We passaged the strain containing the helper plasmid and the target plasmid expressing the L6 gene in a medium containing gentamicin, and the gentamicin concentration was gradually increased during the passage.

[0092] We started with a gentamicin concentration of 1 mg / L, screened for mutants that could grow at high gentamicin concentrations, and gradually increased the gentamicin concentration during the growth cycle. We observed that cells carrying the wild-type L6 gene could not grow in the presence of 100 μg / mL gentamicin, while cells evolved using ITMU could survive in the presence of 300 μg / mL gentamicin ( Figure 6 ). Subsequently, we isolated single colonies from four cell populations that could grow at a gentamicin concentration of 300 μg / mL for sequencing and identified all synonymous mutations in the L6 gene. Using the AlphaFold2 model and AutoDock Vina of the L6 protein to predict the binding site of gentamicin, we found some mutations located near the substrate binding site (such as T67, T68, L71, I121, I133, and I141) ( Figure 7) These mutations may reduce the binding ability of L6 to gentamicin, thereby conferring gentamicin resistance. This method of discovering genotypes that confer antibiotic resistance is generally valuable for improving the effective use of antibiotics.

[0093] The electrotransformation method of Pseudomonas putida KT2440:

[0094] 1) Transfer from the glycerol tube of preserved Pseudomonas putida KT2440 to a test tube containing 5 mL of LB medium at an inoculation amount of 1%, and culture overnight with shaking at 30 °C and 220 rpm on a shaker.

[0095] 2) Transfer to a 300 mL Erlenmeyer flask containing 50 mL of LB medium at an inoculation amount of 1%, add 2 g / L arabinose, and culture with shaking at 30 °C and 220 rpm for 2 - 3 hours.

[0096] 3) Cultivate the bacterial solution until the OD600 reaches 0.6 - 0.8. Transfer 1.5 mL of the bacterial solution to a 1.5 mL sterilized centrifuge tube, centrifuge at 12000 rpm for 1 minute, discard the supernatant, and repeat three times.

[0097] 4) Add 1 mL of 10% glycerol to the centrifuge tube, pipette to mix evenly, centrifuge at 12000 rpm for 1 minute, discard the supernatant, and repeat three times.

[0098] 5) Resuspend the cells with 100 μL of electrotransformation buffer, add the fragment or plasmid to be transformed, and let it stand for 10 minutes.

[0099] 6) Transfer the above - mixed competent cells to a 2 mm electroporation cuvette, use an electroporator to perform electroporation at 1200 V, 25 μF, and 200 Ω. The electroporation constant is about 5.0. Add 1 mL of SOC medium, recover and culture at 30 °C for 1 hour, spread on a solid LB plate containing 25 μg / mL tetracycline, incubate at 30 °C. After single colonies grow on the plate, perform colony PCR verification with corresponding primers, and then perform sequencing verification.

[0100] The method for the evolution of Pseudomonas to tolerate gentamicin:

[0101] Inoculate a single colony of KT2440 containing the helper plasmid and the target plasmid into a 24-well plate containing 2 mL of LB medium. Transfer the bacterial solution every 24 h, and at the same time, increase the gentamicin concentration from 1 μg / mL to 300 μg / mL. After 9 rounds, isolate single colonies and sequence them to search for mutation sites. Construct the wild-type L6 protein and mutants into the pBBR1-MCS2 plasmid and transform Pseudomonas putida KT2440. Inoculate single colonies into a 24-well plate containing 2 mL of LB medium and 100 μg / mL gentamicin. Use a multi-well plate reader (Synergy HT, Biotek, USA) to detect the OD of the bacterial solution 600 。

[0102] (2) Evolution of the lycopene biosynthetic pathway in Corynebacterium glutamicum

[0103] Lycopene is a carotenoid with high commercial value and is widely used in the pharmaceutical, food, and cosmetic industries due to its antioxidant, anti-inflammatory, and anticancer properties. Corynebacterium glutamicum (C. glutamicum), a generally recognized as safe (GRAS) microorganism, has been successfully used for the production of amino acids, vitamins, terpenoids, and biofuels due to its excellent sugar consumption rate under both aerobic and anaerobic conditions, as well as its remarkable tolerance to various chemicals and osmotic stress. Notably, this Gram-positive bacterium contains the crtE, crtB, and crtI genes and is capable of naturally synthesizing lycopene from the precursors of the methylerythritol phosphate (MEP) pathway. These characteristics make C. glutamicum a promising microbial chassis for industrial lycopene production.

[0104] We selected Corynebacterium glutamicum ATCC 13032ΔcrtEb as the host strain. The crtEb gene encodes a lycopene elongase, which is responsible for converting lycopene into flavoxanthin, limiting the accumulation of lycopene. We inserted three homologous lycopene biosynthesis genes, namely geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB), and phytoene desaturase (CrtI), into the target plasmid. After co-transforming the target plasmid and the mutant plasmid into Corynebacterium glutamicum ATCC 13032ΔcrtEb cells, continuous evolution was initiated in 24-well plates containing CGXII medium supplemented with 20 g / L glucose and 2 mM IPTG. After 4 subcultures over 8 days, a distinct color change from pink to red was observed in the culture. We selected the control strain and the evolved strain to grow in CGXII medium to extract lycopene, and then quantified the lycopene yield by high-performance liquid chromatography (HPLC) and studied the correlation between lycopene content and color. Notably, among the five evolved strains, two strains had a significant increase in lycopene yield. The strain with the highest yield had a 3.17-fold increase in lycopene yield compared to the original non-mutagenized strain, reaching 45.9 mg / L( Figure 8 ).

[0105] The electrotransformation method of Corynebacterium glutamicum is as follows:

[0106] 1) Inoculate 3% (v / v) of the overnight culture into 100 ml of BHIS and incubate at 30 °C until mid-exponential phase (OD600 is 0.9).

[0107] 2) Centrifuge the cells and wash 4 times with 10% glycerol (V / V).

[0108] 3) Resuspend in 500 μL of 10% glycerol (V / V).

[0109] 4) Take 100 μL of the cells and add them to a pre-chilled 1.5 mL EP tube. Add the plasmid DNA to the suspension, incubate on ice for 10 min, then transfer to a 0.1 cm electroporation cuvette (Bio-Rad) and electroporate using an electroporator (Bio-Rad, 1800 V, 25 μF, 400 Ω).

[0110] 5) After electroporation, add 1 mL of pre-warmed BHIS culture medium at 46 °C, incubate at 46 °C for 6 min, and then at 30 °C for 2 h. Then spread the cells on an LB agar plate containing antibiotics. After single colonies grow on the plate, verify by colony PCR using the corresponding primers and then by sequencing.

[0111] The above BHIS medium is: 18.5 g / L brain heart infusion broth, 2.5 g / L yeast extract, 5 g / L peptone, 5 g / L NaCl, 91 g / L sorbitol

[0112] The evolutionary method for the production of lycopene by Corynebacterium glutamicum: Both the mutant plasmid and the targeting plasmid are transformed into Corynebacterium glutamicum. The transformed strain is inoculated into a 24-well plate containing 2 mL of CGXII medium (supplemented with 20 g / L glucose and 2 mmol IPTG), and cultured at 30 °C with subculture every 48 hours. After four rounds of subculture, the lycopene production of each strain is measured. 1 mL of the cell culture is centrifuged at 10,000×g for 15 minutes, and the precipitate is washed with deionized water. The pigment is extracted with 10 mL of a methanol:acetone mixture (7:3) at 60 °C for 80 minutes, with thorough vortexing every 20 minutes. The supernatant is quantitatively determined using a Shimadzu high-performance liquid chromatography (HPLC) equipped with a variable wavelength detector (set at 480 nm) and a Symmetry C18 chromatographic column (250 mm × 4.6 mm, 5 μm). Methanol / acetonitrile / dichloromethane (21:21:8) is used as the mobile phase at a flow rate of 1 mL / min, and the column temperature is maintained at 30 °C.

[0113] (3) Evolution of xylose isomerase in Yarrowia lipolytica

[0114] As an unconventional yeast, Yarrowia lipolytica exhibits several key characteristics crucial for industrial-scale production, including biosafety, metabolic robustness, efficient genetic engineering capabilities, and enhanced acetyl-CoA flux. These characteristics make it a highly promising microbial chassis for the synthesis of high-value compounds through industrial fermentation. However, this yeast lacks active xylose metabolism ability, fundamentally limiting its application in the production of cellulosic biofuels and chemicals. Microbial systems capable of actively expressing D-xylose reductase-xylitol dehydrogenase (XR-XDH) and / or xylose isomerase (XI) can effectively convert xylose into xylulose. This intermediate product is subsequently phosphorylated by xylulokinase (XK) to xylulose-5-phosphate, which then enters the pentose phosphate pathway (PPP) to participate in central carbon metabolism. Since XR consumes NADPH while XDH produces NADH, the resulting cofactor imbalance promotes the accumulation of the byproduct xylitol, thereby reducing the metabolic flux of the downstream metabolic pathway. Therefore, cofactor-independent xylose isomerase has become the most promising enzyme for optimizing xylose assimilation.

[0115] We selected PirXI of Piromyces sp. E2 as the target protein. To enhance the xylose metabolism ability of Yarrowia lipolytica mediated by PirXI, we performed evolutionary culturing on the Yarrowia lipolytica PO1f strain containing both the target plasmid and the mutant plasmid in yeast nitrogen base medium (YNB) with 19.75 g / L xylose and 0.25 g / L glucose as carbon sources. After 144 hours of continuous four - passage culturing, the evolved strain gradually showed a growth advantage. After seven - passage culturing, the growth of the evolved strain became stable. Compared with the control strain, the maximum biomass of the evolved strain increased by 49.6%( Figure 9 ). Through plate separation and Sanger sequencing, six synonymous mutants (E15G, L43P, T142A, V187T, W189R, and A431S) were screened out. Under the same culture conditions, the xylose consumption of the strains carrying V187T and W189R mutations reached 3.6 g / L and 2.7 g / L respectively within 48 hours, which were 1.8 - fold and 1.37 - fold higher than that of the wild - type strain respectively( Figure 10 ). Structural analysis showed that the V187 and W189 residues were located near the active site of PirXI, indicating that these substitutions triggered allosteric activation( Figure 11 ).

[0116] The electro - transformation method of Yarrowia lipolytica:

[0117] 1) Inoculate a single colony of the yeast strain for transformation into 5 mL of YPD medium and culture overnight at 30 °C until saturated.

[0118] 2) The night before transformation, inoculate an appropriate amount of the overnight culture into a 300 - mL sterile flask containing 50 mL of YPD medium and shake vigorously at 30 °C until the cell density reaches 1×108 (OD600 is about 1.3 - 1.5).

[0119] 3) Transfer the bacterial liquid into a 100 - mL centrifuge tube and centrifuge at 4 °C at 4000 g to harvest the cultured cells.

[0120] 4) Place the precipitate on ice and resuspend the cells in 16 mL of sterile water. To increase the cell's susceptibility to electroporation, add 2 mL of 10×TE buffer with pH 7.5, shake well, then add 2 mL of 10× lithium acetate, rotate and shake well, and gently shake at 30 °C for 45 min.

[0121] 5) Add 0.5 mL of 1 mol / L DTT and rotate and shake simultaneously, and gently shake at 30 °C for 15 min.

[0122] 6) Add ice-cold sterile water to a total volume of 100 ml, mix well, and centrifuge at 4000×g for 10 min at 4 °C. Add 5 mL of ice-cold 1 M sorbitol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 °C, collect the cell pellet, and add 0.1 mL of sorbitol to resuspend the cells.

[0123] 8) Electroporation: Add 40 μL of yeast cells and 8 μl of plasmid to a sterile ice-cold microcentrifuge tube, and mix well. Transfer to a cold electroporation cuvette, and perform electroporation at 1500 V using a 2 mm electroporation cuvette.

[0124] 9) Add 1 mL of ice-cold 1 mol / L sorbitol to the electroporation cuvette, and gently pipette up and down to mix well.

[0125] 10) Spread directly onto a sorbitol selection medium plate, and incubate at 30 °C until colonies appear on the plate.

[0126] The above YPD medium is: 10 g / L yeast extract, 20 g / L peptone, 22 g / L glucose monohydrate

[0127] The method for evolving xylose isomerase in Yarrowia lipolytica:

[0128] Inoculate a single colony of Po1f containing the helper plasmid and the target plasmid into a test tube containing 2 mL of YNB medium. Transfer the bacterial solution every 48 h. After seven subcultures, individual colonies are isolated and sequenced to detect mutations. The wild-type xylose isomerase and mutants are constructed into the plasmid Ylep-leu-XK expressing xylokinase, and transformed into Yarrowia lipolytica. Inoculate a single colony into a shake flask containing 50 mL of YNB medium, 19.75 g / L xylose, and 0.25 g / L glucose. Each experiment is repeated three times. Measure OD600 using a spectrophotometer (Shimazu, Japan). The fermentation samples are centrifuged at 10000×g for 5 min, and the supernatant is taken to detect extracellular metabolites. Quantitative determination of xylose is performed using HPLC (Shimadzu, Japan), equipped with a refractive index detector (RID-10A, Shimadzu, Japan) and an Aminex HPX-87H ion exchange column (Bio-Rad, USA).

[0129] The above results show that ITMU not only achieves targeted mutagenesis in model organisms, but also exhibits good applicability in non-model prokaryotic and eukaryotic microorganisms.

[0130] Matters not covered by this invention are well-known technologies.

[0131] The amino acid sequence of PolI4MT

[0132]

[0133] Nucleotide sequence of PolI4MT

[0134]

[0135] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or make equivalent substitutions to the technical solution of the present invention as needed, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A multi-host in vivo continuous evolution system, characterized in that, The system includes plasmids, with at least two plasmids, including at least an auxiliary plasmid and a target plasmid; The auxiliary plasmid contains the coding gene of the fusion protein TadA8e-RepA of RepA helicase and deaminase TadA8e, the coding gene of the initiator protein RepC, and the coding gene of the fusion protein RepB-PolI of primase and error-prone PolI; The target plasmid contains the coding gene of the target gene and the replicon OriV.

2. The multi-host in vivo continuous evolution system according to claim 1, wherein The error-prone PolI is a variant of DNA polymerase I derived from Escherichia coli, and the variant contains mutations of D424A, I709N, R751K, and A759R based on wild-type DNA polymerase I; Furthermore, the error-prone PolI is a variant of DNA polymerase I inserted with thioredoxin.

3. The multi-host in vivo continuous evolution system according to claim 1, characterized in that The target gene is the coding gene and / or non-coding gene of one or more proteins; Furthermore, the target gene includes the resistance gene of antibiotics, the coding gene of the enzyme in the catabolic pathway, the coding gene of the enzyme in the anabolic pathway, the coding gene of the DNA-binding protein, the coding gene of the nuclease, the coding gene of the glycosidase, and the coding gene of the protease.

4. The orthogonal multi-host in vivo continuous evolution system according to any one of claims 1-3, characterized in that, Except for the coding genes of the target gene and the error-prone PolI, the coding genes of the above other enzymes or proteins are all derived from broad-host plasmids, and the broad-host plasmids include plasmids of IncP, IncN, IncQ, and IncW groups. Furthermore, the broad-host plasmid is RSF1010.

5. A method for an orthogonal multi-host in vivo continuous evolution system, characterized in that, The method includes transforming the system according to any one of claims 1-4 into a host cell to achieve continuous evolution of the target protein in the host cell in vivo.

6. The method according to claim 5, wherein The method includes: using the initiator protein RepC in the broad-host plasmid replication system to bind to the replicon OriV of the plasmid to promote double-strand separation; using the DNA helicase RepA to unwind the double-stranded DNA in two directions, and the deaminase TadA8e to deaminate the single-stranded DNA; using the primase RepB fused with error-prone PolI to recognize two initiation signals inside OriV and synthesize primers; after the primers are synthesized, RepB separates from the replicon, and PolI binds to the primers to start replicating the plasmid and introduce mutations into the plasmid, thereby achieving continuous evolution of the target protein in the host cell in vivo.

7. The method according to claim 6, characterized in that, The host cell includes prokaryotic cells and eukaryotic cells.

8. The method according to claim 7, wherein The prokaryotes include bacteria, and the eukaryotes include fungi.

9. The method according to claim 8, characterized in that, The bacteria are Gram-positive bacteria or Gram-negative bacteria, including Escherichia coli, Corynebacterium glutamicum, and Pseudomonas putida. The fungus is Yarrowia lipolytica.

10. The application of the system according to any one of claims 1-4 and / or the method according to any one of claims 5-9 in protein directed evolution.

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