WO2023164788A1 - Procédé de criblage à haut débit de lantibiotiques - Google Patents

Procédé de criblage à haut débit de lantibiotiques Download PDF

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WO2023164788A1
WO2023164788A1 PCT/CN2022/078528 CN2022078528W WO2023164788A1 WO 2023164788 A1 WO2023164788 A1 WO 2023164788A1 CN 2022078528 W CN2022078528 W CN 2022078528W WO 2023164788 A1 WO2023164788 A1 WO 2023164788A1
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mutant
automated
library
activity
plasmid
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PCT/CN2022/078528
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Chinese (zh)
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司同
郭二鹏
张雅婷
付立豪
张建志
陈永灿
张智彧
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中国科学院深圳理工大学(筹)
深圳先进技术研究院
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Priority to PCT/CN2022/078528 priority Critical patent/WO2023164788A1/fr
Publication of WO2023164788A1 publication Critical patent/WO2023164788A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • the invention relates to a high-throughput screening method for lantibiotics, in particular to a method for screening lantibiotics based on an automated platform.
  • Nisin has been used as a commercial food preservative for more than 70 years, and there is still no multi-drug resistance.
  • the common feature of lantibiotics is that the molecular structure contains one or more thioether-linked lanthionine (Lan) and/or methylated lanthionine (MeLan).
  • lanthipeptides are generally divided into 4 main subtypes. Among them, the synthetase of type I lanthipeptide is dehydrated and cyclized by dehydratase LanB and cyclase LanC, respectively; while the synthetase of type II lanthipeptide LanM is a bifunctional enzyme with both dehydration and cyclization. functional domain.
  • the N-terminal dehydration region of LanM has no homology with other lanthipeptide synthases, while the C-terminal cyclization domain is similar to the C-terminal cyclization domains of type I lanthipeptide synthase LanC and type IV lanthipeptide synthase LanL They are homologous and contain a conserved zinc ion binding site.
  • the synthetase LanKC of type III lanthipeptide and the synthetase LanL of type IV lanthipeptide have three functional domains, and the N-terminus contains two lyases (lyase) and kinase (kinase).
  • Structural and functional domains that act together in the dehydration reactions of Ser and Thr, in which the C-terminal cyclase of the type III lanthipeptide synthetase LanKC has homology with the other three types of cyclization domains, but does not contain the conserved zinc ion binding site.
  • Type I lanthipeptide synthase uses glutamylated tRNA to activate the hydroxyl group of Ser/Thr for dehydration, while type II, type III and IV lanthipeptide synthase dehydration is based on phosphorylation modification.
  • the lanthipeptide gene forms a precursor peptide through transcription and translation, and then, the N-terminal leader peptide/or C-terminal follower peptide (follower peptide) of the precursor peptide is recognized by lanthipeptide synthetase peptide), and introduce dehydration and cyclization modifications in the core peptide; subsequently, the leader peptide is removed by the protease domain of the protease or transporter, releasing the functionally active mature lanthipeptide.
  • modifying enzymes that can introduce chemical modifications such as hydroxylation, methylation, glycosylation, halogenation, acylation, and epimerization. These modifications can improve the stability of lanthipeptide under conditions such as high temperature, non-physiological pH, and protease hydrolysis, or affect its biological activity.
  • the main antibacterial mechanism of lantibiotics is to combine with Lipid II, the precursor of bacterial cell wall synthesis. On the one hand, it prevents the biosynthesis of cell wall, and on the other hand, it perforates the cell membrane to cause the outflow of cell components to achieve antibacterial effect.
  • Lantibiotics show strong activity against Gram-positive bacteria, and also have a good inhibitory effect on some multidrug-resistant bacteria, such as drug-resistant Staphylococcus aureus (methicillin resistant Staphylococcus aureus, MRSA), vancomycin-resistant intestinal Streptococcus (vancomycin resistant enterococci, VRE), Streptococcus pneumoniae (Streptococcus pneumoniae) and Clostridium difficile (Clostridium difficile), etc.
  • the precursor peptide of lanthipeptide is encoded by a gene, which can quickly produce structurally diverse products by introducing mutations. Meanwhile, the lanthipeptide precursor peptide has a modular composition.
  • the lanthipeptide biosynthetic gene cluster of Prochlorococcus contains a lanthipeptide synthetase ProcM and about 30 precursor peptide encoding genes procA.
  • leader peptide The sequence of the leader peptide is highly conserved, while the core peptide sequence is highly variable. Change.
  • other lanthipeptide core peptides were fused with nisin leader peptides to obtain artificial chimeric precursor peptides that could also be recognized and modified by nisin synthase NisBC.
  • lanthipeptide synthetases including NisBC and ProcM have high substrate promiscuity. Combining these characteristics, researchers have developed various new methods based on the principles of modularization and "plug and play" to carry out large-scale engineering transformation and mining for the biosynthesis of lanthipeptide antibiotics. While deeply understanding its structure-activity relationship, biosynthetic process, active target and mechanism, the pharmaceutical properties of lanthipeptide antibiotics can be improved, and even new activities and applications can be developed.
  • the genetic manipulation of most lanthipeptide antibiotics is very difficult.
  • the genetic manipulation of engineering transformation is usually carried out in Escherichia coli or Lactobacillus.
  • the expression modification of lantibiotics and the release of the core peptide were carried out separately, and the introduction of various commercial enzyme cutting sites also made the removal of the leader peptide not challenging.
  • screening platforms such as microfluidic systems, microdroplet array systems, cell-free systems, and micro-nanoreactors have been used for high-throughput screening of lantibiotic mutants, but the expression modification and guided peptide transfer of lantibiotics The step-by-step process of elimination hinders the scale and throughput of mutant screening to some extent.
  • An object of the present invention is to provide a high-throughput automatic screening method for lantibiotics.
  • Another object of the present invention is to provide the lantibiotics obtained by screening.
  • Another object of the present invention is to provide the application of the screening method in the study of the activity-structure relationship of lantibiotics, so as to facilitate the subsequent rational design of lantibiotics with better activity.
  • the present invention provides a high-throughput screening method for lantibiotics, the method comprising:
  • the antibacterial activity characterization includes obtaining the antibacterial activity of each mutant by an antibacterial method to achieve high-throughput automated lantibiotic activity screening, so
  • the above sample detection includes high-throughput automated MALDI-TOF detection of target products.
  • the process of constructing a lantibiotic site-directed saturation mutation library includes:
  • Gibson assembly was performed on the PCR product using an automated liquid pipetting workstation and an automated PCR machine to obtain a saturated mutant plasmid library.
  • the automated high-throughput construction and screening process of the site-directed saturation mutation library can avoid the high-intensity screening process of the experimenter, and at the same time avoid human errors caused by manual operations, and can accurately and efficiently complete the construction, transformation, and clone picking of the site-directed saturation mutation library and rearrangement after library screening.
  • the process of performing site-directed saturation mutation PCR includes:
  • the workstation will mix the components in the reaction system into the PCR reaction plate according to the automatic PCR amplification reaction program, and transfer them to the automatic PCR reaction plate through the automatic pipetting arm.
  • PCR reaction in the instrument after the reaction, the automated robot arm will transport it back to the automatic pipetting workstation, add DpnI enzyme, and then carry out the reaction in the automatic PCR instrument to digest the template plasmid;
  • fragments are amplified by PCR.
  • Fragment 1 is amplified by the mutated forward primer and the kanamycin-resistant reverse primer on the carrier through reaction system 1.
  • Fragment 2 is amplified by the mutated reverse primer and kanamycin-resistant reverse primer.
  • the resistant forward primer pRSF-Kana-F was amplified through reaction system 2.
  • the process of performing Gibson assembly includes:
  • the automated liquid pipetting workstation mixes the groups in the Gibson assembly reaction according to the automated Gibson assembly program and sends them to the PCR machine for the reaction through the automated robotic arm. Assemble and obtain a saturated mutant plasmid library;
  • each gene numbering site saturation mutation library is obtained by Gbsion assembly of two fragments amplified using the recombinant plasmid as a template.
  • the process of extracting plasmids and transforming expression strains includes:
  • Escherichia coli competent cell is Escherichia coli Dh5a competent cell; Transformation process comprises:
  • the high-throughput screening method for lantibiotics of the present invention further includes:
  • the process of extracting plasmids and transforming expression strains also includes:
  • the process of transforming into the expression strain BL21(DE3) includes: providing BL21(DE3) competent cells containing pBAD18-SpompA-LicP-25-433/SRRz, using a liquid transfer station to aliquot the competent cells at 4°C In a 96-well PCR tube on the plate; add the mutant library plasmids to the competent 96-well plate, incubate, transfer the 96-well plate to the heating module for heat shock using the robotic arm, and transfer the competent to the supplemented medium using the 96-channel MCA
  • Use the robotic arm to transfer the 96-well deep-well plate to the incubator for culture; use the robotic arm to transfer the cultured 96-well plate to the pipetting workstation again, and use the 96-flux MCA to extract the transformed bacterial droplets Add to Petri dishes and incubate overnight.
  • the high-throughput screening method for lantibiotics of the present invention may further include the process of performing sequencing verification on a saturated mutant library.
  • the process of using an automated platform to characterize the antibacterial activity and sample detection of all mutants includes:
  • the specific operation is as follows: put the preserved mutant strain into the pipetting workstation, use the robotic arm of the automatic function island to grab the strain and place it in the film tearing instrument, and put the single sealing film on the preserved 96-well plate Uncover, then use the MCA96 channel to dip the colony in the 96-well seed preservation plate, inoculate it into the 96-well plate added with LB, and culture the inoculated 96-well plate overnight through the robotic arm of the functional island; Take out the cultured mutant library from the box and place it in the pipetting workstation.
  • the process of using an automated platform to characterize the antibacterial activity of all mutants includes:
  • a self-cleaving lantibiotic expression system was constructed, and the expression bacteria were self-lysed to perform antibacterial experiments to characterize their activity.
  • the process of using an automated platform to characterize the antibacterial activity of all mutants includes:
  • Lactococcus lactis HP was used as a sensitive indicator bacterium for activity testing, and antibacterial experiments were carried out to characterize the activity.
  • expression can also be identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
  • the present invention is to use the liquid transfer workstation to automatically transform the plasmid of the mutant library into the competent state of BL21 (DE3) containing the pBAD18duet-OmpA-LicP-SRRz plasmid, and use Qpix to pick single Clones were cloned into a 96-well deep-well plate, transferred to an automated incubator for overnight culture, and then preserved using a Tecan pipetting workstation. At the same time, use the pipetting workstation to inoculate and culture again. When the OD600 is measured between 0.6 and 0.8 by using the pipetting workstation and microplate reader, add IPTG and Arabinose to induce in the automatic incubator.
  • a dual-plasmid expression system is used in the present invention to construct a self-cleavage system.
  • the double plasmid was transformed into E.coli BL21(DE3) and cultured under the corresponding temperature conditions, the expression was induced, and the guide peptide was removed to obtain mature lanthipeptide.
  • Lactococcus lactis HP was used as a sensitive indicator bacterium for activity testing, and the antibacterial activity was evaluated by comparing the size of the inhibition zone of each mutant of lanthipeptide.
  • the present invention can complete the expression modification of lantibiotics and the removal of guide peptides to release mature peptides in the same cell, which solves the key experimental steps for the release of mature lantibiotics, and obtains mature lantibiotics more conveniently and efficiently.
  • Lanthipeptide is a group consisting of progenitors, progenitors, and progenitors, progenitors, and progenitors, and obtained lantibiotics more conveniently and efficiently.
  • the process of using an automated platform to perform sample detection on all mutants includes high-throughput activity screening of lantibiotics:
  • the mutant expression library is placed on the carrier of the automation platform, and the mutant library is transferred to the film tearing instrument by the robotic arm, and the parafilm is taken out; the robotic arm transfers the mutant library seed solution to the pipetting workstation for inoculation, and then transferred to the incubator Cultivate overnight to allow each mutant to reach saturation;
  • the cultured mutant library was inoculated with a pipetting workstation for secondary expansion, and the OD was between 0.6-0.8 to induce expression;
  • the expressed mutant library is transferred to a centrifuge by a robotic arm and centrifuged to remove the supernatant;
  • the lysed bacteria are heated to completely kill the remaining surviving E.coli;
  • mutant library was centrifuged, and the supernatant was extracted using a 96-channel MCA, and added to the prepared agar antibacterial plate, and then the robotic arm was used to transfer the antibacterial plate to the incubator for overnight culture;
  • the diameter of the wild-type bacteriostatic zone at each mutation site is used as a standard, and the length of the mutant bacteriostatic zone is measured compared with the wild-type to obtain a complete bacteriostatic activity spectrum. active screening.
  • the process of using an automated platform to perform sample detection on all mutants includes high-throughput MALDI-TOF MS identification of lantibiotics.
  • the establishment of a high-throughput automated MALDI-TOF MS method for the detection of lantibiotics has achieved ultra-high-throughput sample analysis. Compared with the traditional chromatography-mass spectrometry detection method, it is shortened from about 30 minutes per sample to each Sample 5s, greatly improving the experimental throughput.
  • the experiment from culture expression to sample detection is through an automated workstation, which reduces human errors and speeds up the experimental process.
  • the high-throughput MALDI-TOF MS identification process includes:
  • the lantibiotic mutant expression library engineered to add the LicP enzyme cleavage site was cultivated overnight, and the samples were extracted using 96-channel MCA, and inoculated into polyvinylidene fluoride (Poly( vinylidene fluoride), PVDF) membrane, overnight culture;
  • the colony after induced expression is transferred to the incubator together with the culture plate for incubation, and the colony is fully lysed so that the LicP enzyme and the modified mutant come into contact with each other to release the mature mutant;
  • the high-throughput screening method for lantibiotics of the present invention is suitable for the expression and activity detection of various lantibiotics, for example, the lantibiotics may include one of haloduracin ⁇ chain and Nisin or more.
  • the present invention also provides a lantibiotic, which is screened according to the method of the present invention.
  • the lantibiotics include: one or more of halA1-A4H, A15H, etc.
  • the present invention also provides the application of the screening method in the research on the structure and activity of haloduracin ⁇ .
  • the study on the structure and activity of haloduracin ⁇ includes studying the relationship between the amino acid position of lanthipeptide and the activity of the lanthipeptide, for example, in some specific embodiments of the present invention, the G11, N12, G14, E22 of haloduracin ⁇ are studied The relationship between amino acid sites such as , 24M and/or P25 and the activity of the lanthipeptide.
  • the present invention conducts experimental tests through the two-component lanthipeptide antibiotic haloduracin ⁇ chain, and successfully obtains multiple site-directed saturation mutation libraries at non-structural key sites of halA1 through a high-throughput automation platform, and utilizes The automated platform performs antibacterial activity characterization and sample testing on all mutants.
  • the expression of each mutant was successfully detected by high-throughput MALDI-TOF MS, and the antibacterial activity of each mutant was simultaneously obtained by the agar plate antibacterial method, and high-quality data with high stability and no artificial experimental errors were obtained, successfully revealing The structure-activity relationship of hal ⁇ was determined, providing reliable data support for the subsequent design of new lanthipeptide antibiotics.
  • cell compartmentation is skillfully used to localize the guiding peptide-cleaving protease LicP to the periplasmic space, and the lanthipeptide precursor and its modifying enzyme are co-expressed in the cytoplasm to obtain the modified lanthipeptide.
  • Subsequent temperature control of phage cleavage gene expression and temporal control directs the peptidic cleaving enzyme to contact the modified lanthipeptide, releasing the mature lanthipeptide.
  • this protocol enabled the production of mature lanthipeptide in a single clone.
  • the high-throughput automated screening platform is a functional island that integrates different functional components.
  • the robotic arm is used to connect the various components in series to form an automated workflow to realize library construction, transformation, clone picking, seed preservation, inoculation and culture, and plasmid extraction.
  • Activity characterization and mass spectrometry identification and other processes are mechanically automated. Every link in the automated workflow is adjusted on the basis of the existing development plan, and based on the combination of different processes of a single step, the corresponding experimental results are obtained.
  • the present invention uses an automated workstation to realize the construction and screening of a high-throughput automated site-directed saturation mutation library for lanthipeptide antibiotics, then heterologously express mature lanthipeptide antibiotics based on the E. coli self-lysis system, and finally through automated The workstation automates high-throughput product identification and activity characterization.
  • This system is not only suitable for the engineering transformation of existing lanthipeptide antibiotics, but also obtains its structure-activity relationship, which is of guiding significance for the transformation and obtaining of highly active lanthipeptide antibiotics; Activity screening of antibiotics to excavate new lanthipeptide antibiotics. Therefore, the application of this automated high-throughput activity screening platform to the transformation and mining of highly active lanthipeptide antibiotics has far-reaching significance for solving the global problem of drug-resistant bacteria.
  • Fig. 1 is a schematic flow chart of high-throughput antibacterial activity screening and mass spectrometry detection of the lanthipeptide antibiotic of the present invention.
  • Fig. 2 is a schematic diagram of the sorting process of the mutant library of the present invention.
  • Figure 3 shows the sequencing results of the site-directed saturation mutation library in Example 1 of the present invention.
  • Figure 4 shows the biosynthetic pathway of Hal ⁇ engineered in the present invention.
  • FIG. 5 is a schematic diagram of the production of mature lanthipeptide by E.coli.
  • the upper left picture shows that the LicP enzyme is expressed in the periplasmic space, and LanA and LanM are expressed in the cytoplasm to form the modified lanthipeptide mLanA; the upper right picture shows that after E.coli cleavage, the LicP enzyme recognizes the cleavage sequence and releases the mature lanthipeptide.
  • Figure 6 shows the results of the analysis of mature hal ⁇ expression in Escherichia coli self-lysed by agar inhibition test and MALDI-TOF MS.
  • Figure 7 shows the antibacterial screening results and mass spectrometric identification results of HalA1-12, 16, and 19 libraries.
  • Figure 8 is a heat map of activity screening of hal ⁇ mutants.
  • Figure 9 is a schematic diagram of the high-throughput MALDI-TOF MS identification workflow.
  • Fig. 10 is a schematic structural diagram of the automation function island of the present invention.
  • 11A to 11C are schematic diagrams of the transformation of the nisin biosynthesis pathway, activity measurement and mass spectrometry detection results of the present invention.
  • the instruments, equipment, materials, and biochemical reagents used are all conventional commercially available reagents, and the technical means used are conventional means familiar to those skilled in the art, or operated according to the conditions suggested by the instrument manufacturer .
  • this embodiment provides a high-throughput automated screening method for lantibiotics, the main process of the method includes: automatic construction and screening of a site-directed saturation mutation library, automatic extraction of plasmids and transformation of expression strains, and then Perform automated high-throughput antibacterial activity characterization and high-throughput product identification.
  • the high-throughput automated activity screening of lantibiotics in this embodiment is to screen the activity of the two-component lantibiotic haloduracin ⁇ chain, and the specific method includes steps:
  • each gene number site saturation mutation library is obtained by Gbsion assembly of 2 fragments amplified with recombinant plasmids as templates
  • Fragment 1 (PCR-1) was obtained by amplifying the mutant forward primer (halA1-F) and the kanamycin-resistant reverse primer (pRSF-Kana-R) on the vector through reaction system 1
  • fragment 2 (PCR-2 ) was amplified by the mutation reverse primer (halA1-R) and the kanamycin resistance forward primer pRSF-Kana-F through reaction system 2.
  • a saturation mutation library was constructed for a total of 20 sites numbered 2-6, 9-16, 19, 21-22, 24-26, and 28 in the gene halA1 sequence. 2 also has 20 pieces, which are assembled in two pieces in one-to-one correspondence with the pieces.
  • Fragment amplification reaction system 1 is 1 ⁇ l of pRSFDuet-halA1-halM1 plasmid (concentration 1ng/ ⁇ l), 2 ⁇ l of forward mutation primer (10 mM), 2 ⁇ l of reverse primer (10 mM), 25 ⁇ l of PrimeSTAR premixed high-fidelity polymerase (purchased from Takara ), double distilled water 20 ⁇ l, and the total reaction volume was 50 ⁇ l.
  • the recombinant plasmid pRSFDuet-halA1-halM1 is the combination of haloduracinA1 (halA1, length 210bp, sequence shown in SEQ ID No.1) and its modification enzyme haloduracin M1 (halM1, length 3180bp, sequence shown in SEQ ID No.2)
  • haloduracinA1 halA1, length 210bp, sequence shown in SEQ ID No.1
  • halM1 halM1, length 3180bp, sequence shown in SEQ ID No.2
  • the recombinant plasmid with a total length of 7202bp was constructed on the pRSFDuet vector.
  • the specific operation can be carried out as follows: put the reagents of the above PCR reaction system in the reagent tanks of the automatic liquid transfer workstation, and the workstation will mix the components in the reaction system into the PCR reaction according to the edited automatic PCR amplification reaction program script.
  • the plate was transferred to the automatic PCR instrument through the automatic pipetting arm.
  • the reaction program was 98°C pre-denaturation for 5 minutes, followed by 30 cycle reactions, each cycle including: 98°C denaturation for 15 s, 53°C annealing for 15 s, and 72°C extension for 10 s , followed by extension at 72°C for 5 min, and finally stored at 4°C.
  • the automated robotic arm transported it back to the automated liquid transfer workstation, added 10U DpnI enzyme (purchased from NEB, USA) to each reaction, and then reacted in an automated PCR instrument at 37°C for 1 hour to digest the template plasmid for purification .
  • 10U DpnI enzyme purchased from NEB, USA
  • HalodurcinA1 gene sequence (SEQ ID No.1):
  • HaloduracinM1 modification enzyme gene sequence (SEQ ID No.2):
  • the PCR product (purified nucleic acid fragment) of the previous step is carried out to Gibson assembly by using an automatic pipetting workstation and an automatic PCR machine, and each reaction assembles a PCR-1 and a PCR-2 into a complete plasmid (That is, a saturated mutant plasmid), according to the design of PCR primers, each pair of different PCR-1 can be assembled with the corresponding PCR-2, and finally a total of 20 saturated mutant plasmid libraries can be obtained by assembling.
  • This process can be carried out simultaneously for 96 an assembly reaction.
  • the Gibson assembly reaction solution was pre-configured before the experiment, including 320 ⁇ l 5X ISO buffer, 0.64 ⁇ l T5 exonuclease (10 U/ ⁇ l, purchased from NEB Company in the United States), 20 ⁇ l Phusion polymerase (2 U/ ⁇ l, purchased from NEB Company in the United States) ), 160 ⁇ l Taq ligase (40 U/ ⁇ l, purchased from NEB Company, USA), double-distilled water to make up to 1.2 mL, and store at -20°C.
  • the specific operation can be carried out as follows: Place the components required for the Gibson assembly reaction into the reagent tank of the automatic pipetting workstation.
  • the reaction system is: Gibson assembly reaction solution 7.5 ⁇ l, purified nucleic acid fragment 1: 1.25 ⁇ l, purified Nucleic acid fragment 2: 1.25 ⁇ l, total reaction system 10 ⁇ l.
  • the automated pipetting workstation mixed the groups in the Gibson assembly reaction according to the edited automated Gibson assembly program script and sent them to the PCR instrument through the automated robotic arm for reaction at 50°C for 1 hour. 20 saturation mutant plasmid libraries (halA1 mutant library plasmids) were obtained by assembly.
  • the above-mentioned saturated mutant plasmid can be transformed into E. coli competent cells using an automated liquid pipetting workstation, and this process can perform 96 transformation experiments at the same time.
  • the transformation of Escherichia coli Dh5a competent cells and expression strain BL21(DE3) was carried out. The specific operation can be carried out as follows:
  • Transformation of Escherichia coli Dh5a competent cells (purchased from Kangti Biology): use the automatic pipetting workstation to add 50 ⁇ l of competent cells to the saturated mutant plasmid according to the edited automated plasmid transformation program script, and Incubate on the 4°C low-temperature control module of the liquid transfer workstation for 30 minutes, transfer to the 45°C heating control module through the mechanical gripper of the workstation, heat shock for 60 seconds, transfer to the 4°C low-temperature control module and incubate for 2 minutes, and use 96-channel MCA to transfer the transformed feelings Add it to 500 ⁇ l of LB medium without antibiotics, then transfer to a 37°C incubator and incubate for 1 hour, centrifuge to remove 300 ⁇ l of the supernatant, and resuspend the remaining 200 ⁇ l of the bacterial solution, evenly drop it into a plate containing antibiotics, and place it at 37 Invert the cultured liquid in the microbial incubator at °C.
  • Transformation of expression strain BL21(DE3) provide BL21(DE3) competent cells containing pBAD18-SpompA-LicP-25-433/SRRz, and use a pipetting workstation to distribute the competent cells into 96-well PCRs placed on a 4°C heating plate tube, 30 ⁇ l per well.
  • the 96-channel MCA transfer competence was added to a 96-well deep-well plate with 500 ⁇ l of anti-LB medium, and the 96-well deep-well plate was transferred to an incubator for 2 hours at 30° C. by using a robotic arm.
  • Construction of the DH5 ⁇ library bacterial clone picking (sorting process as shown in Figure 2): Use the Qpix automatic picking cloner to pick the transformed plate into a 96-well plate for culture, and pick two 96-well plates from each library-transformed DH5 ⁇ plate Plate, after overnight incubation, use an automated pipetting station to remove 100 ⁇ l from each well for sequencing. According to the sequencing results, different amino acid mutants at different positions were selected, rearranged in a 96-well plate according to the alphabetical order of the amino acid abbreviations, and 700 ⁇ l of the mutant culture solution was added to 300 ⁇ l of 50% glycerol, and finally sealed with a sealing device. Stored at -80°C for conservation.
  • this step the success of the construction is evaluated by sequencing the transformed saturated mutation library, and the sequencing results show that there is an NNK nest peak at the position of the target number, which means that the library construction is successful.
  • This step can also be performed before the aforementioned step “automated clone picking", or simultaneously with the aforementioned step “automated clone picking”.
  • the specific operation can be carried out as follows: take out the plate cultivated overnight in the previous step, add 3mL LB medium dropwise on the plate, scrape off all the colonies in the plate with a coating stick, collect them into a new Ep tube, and draw 500 ⁇ l of bacteria from it. mixed with 500 ⁇ l 40% glycerol solution and stored in a -80°C refrigerator, and the remaining bacterial solution was sequenced by a sequencer (or sent to Shanghai Sangon Biotech Co., Ltd. for sequencing). The sequencing results are shown in Figure 3: the corresponding mutation positions Point NNK mutations were successfully obtained mutation library.
  • the robot arm of the automation function island uses the robot arm of the automation function island to grab the strain and place it in the tearing film instrument, remove the single sealing film on the 96-well plate of the preserved species, and then use the MCA96 Dip the colony in the 96-well seed preservation plate, inoculate it into a 96-well plate added with LB, and culture the inoculated 96-well plate overnight at 30°C through the robotic arm of the functional island. Then, the robotic arm takes out the cultured mutant library from the incubator and places it in the pipetting workstation.
  • the workflow of automated high-throughput plasmid extraction is as follows: firstly, the bacteria cultured overnight was centrifuged to remove the supernatant, then added P1 in the plasmid extraction kit to resuspend, added P2 to mix and lyse for 3 minutes, added P3 to terminate the reaction, and centrifuged for 10 minutes to take Add magnetic beads to the supernatant, take the supernatant after shaking, add washing solution and shake to remove the supernatant, repeat washing three times, then add 50 ⁇ l deionized water to wash the plasmid, extract the dissolved plasmid and transfer it to a 96-well plate for sealed storage.
  • the sample was washed three times with a single pipetting workstation on the functional island and a high-speed centrifuge, and finally dissolved in 50ul LB medium for lysis in a 38°C incubator for 4h. Transfer the lysed deep-well plate to a water bath at 80°C for 10 minutes to inactivate E.coli that has not been lysed to prevent E.coli from affecting the experimental results.
  • the workflow for the characterization of lanthipeptide antibiotics is basically a continuous step, including production and modification of lanthionine peptides, removal of guide peptides, biological activity screening, and mass spectrometry analysis. Among them, the production, modification and removal of lanthipeptides are quite mature.
  • the N-terminal natural secretion signal of licP enzyme was replaced by the OmpA signal peptide of E. coli, which localized LicP enzyme to the periplasmic space.
  • Adding a histidine tag to the N-terminus of halA2 avoids the degradation of the engineered halA2 by endogenous E. coli peptidases to a certain extent, and the addition of a histidine tag also facilitates the purification of subsequent mutants.
  • E. coli lytic gene SRRz from phage ⁇ was used.
  • the SRRz gene was controlled by two heat-inducible promoters ⁇ cI857/pR.
  • Escherichia coli containing the self-lysing plasmid can grow normally at 30°C, and when transferred to 38°C for about 4 hours, more than 90% of the cells were lysed.
  • the lytic activity of cells is due to the fact that the products of lytic genes can form holes in the inner membrane of cells, degrade peptidoglycan, and destroy the outer membrane of cells. Lysis of the cells releases LicP in the periplasmic space into contact with the modified halAl, removes the leader peptide, and releases the mature hal ⁇ (Fig. 5).
  • the implementation of the self-lysis system relies on a two-plasmid expression system.
  • his-halA1 and halM1 were assembled into the pRSFDuet binary plasmid, which was induced by IPTG; whereas SPOmpA-licP and SRRz were assembled into the pBAD18duet plasmid, which was induced by arabinose and temperature transformation, respectively.
  • the double plasmid was transformed into E.coli BL21(DE3) and cultured under the corresponding temperature conditions, the expression was induced, and the guide peptide was removed to obtain mature hal ⁇ .
  • Lactococcus lactis HP was used as a sensitive indicator bacterium for activity testing, and the expressed hal ⁇ and hal ⁇ purified from B, halodurans synergistically inhibited the growth of the indicator bacterium, and the antibacterial activity was evaluated by comparing the size of the inhibition zone of each mutant of hal ⁇ , and the matrix was assisted Laser desorption ionization time-of-flight mass spectrometry (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS) was used to identify the expression of hal ⁇ (as shown in Figure 6).
  • MALDI-TOF MS Laser desorption ionization time-of-flight mass spectrometry
  • the mutant expression library is placed on the carrier of the automated platform, the mutant library is transferred to the film tearing instrument by the robotic arm, and the parafilm is removed; the robotic arm transfers the mutant library seed solution to the pipetting workstation for inoculation, and then Transfer to an incubator for overnight culture at 30°C, so that each hal ⁇ mutant can be cultured to saturation.
  • the cultivated mutant library was inoculated with 1% of the liquid transfer station for secondary expansion, about 4 hours, and the OD was between 0.6-0.8, and the final concentration of 1mM IPTG and 10mM arabinose were added to induce expression at 18°C for 20 hours.
  • the expressed mutant library was transferred to a centrifuge at 8000xg for 10 min using a robotic arm, and the supernatant was removed.
  • the heated mutant library was centrifuged at 8000g for 10min, and 10 ⁇ l of the supernatant was extracted using a 96-channel MCA, and added to the prepared agar antibacterial plate. At the same time, 3 ⁇ l of purified 200nM hal ⁇ was added as a positive control, and then the antibacterial plate was transferred by the robotic arm Incubate overnight in a 30°C incubator.
  • the antibacterial results of the library agar are shown in picture A in Figure 7. Taking the diameter of the wild-type inhibition zone at each mutation site as a standard, measure the length of the mutant inhibition zone and compare it with the wild-type to obtain complete antibacterial activity The spectrum is shown in Figure 8.
  • G11, N12 and G14 in the A loop are conserved sequences, and after mutation to any other amino acid, the activity is significantly reduced; E22, 24M and P25 in the B loop are also conservative sequences, and the activity after mutation is relatively low.
  • the wild type is significantly worse; the present invention also finds that the R9 site in the A ring has an antibacterial zone when it is mutated into a cationic amino acid, and its adjacent I6 and L10 are only mutated into several Only non-polar amino acids have the same antibacterial activity as the wild type; the mutation of the 5th site to Cys has residual antibacterial activity, and the mutation of other sites to Cys will cause the loss of halA1 activity, which may be caused by the introduction of Cys. halA1 does not loop properly and is inactivated.
  • the lantibiotic mutant expression library engineered to add the LicP enzyme cleavage site was cultivated overnight, and 3 ⁇ l was extracted using Tecan’s 96-channel MCA, and inoculated onto the polyvinylidene fluoride membrane placed on the square culture dish of solid LB containing antibiotics (polyvinylidene fluoride, PVDF), culture overnight at 30°C. After the colony grew up, the PVDF membrane with the colony was manually transferred to a solid LB square petri dish containing IPTG and arabinose, and induced at 18°C for 20h.
  • the induced expression colony together with the culture plate was transferred to a 38°C incubator with a robotic arm and incubated for 4 hours, and the colony was fully lysed so that the LicP enzyme and the modified halA1 mutant came into contact with each other to release the mature hal ⁇ .
  • CHCA ⁇ -cyano-4- Hydroxycinnamic acid
  • the above-mentioned method of this embodiment is preferably completed by using an automated functional island.
  • an automated functional island Please refer to FIG. Sealing machine, film tearing machine, microplate reader, and automated shaking incubator, among which Evoware, the operating system of the automated liquid transfer workstation, is provided by the manufacturer, and Momentum, the operating system of the functional island, is provided by Thermo Fisher.
  • the automatic pipetting workstation is equipped with flexible eight-channel pipetting tips, high-throughput 96-channel pipetting tips, multi-functional gripper robotic arm, low temperature control module, heating control module, oscillation module, orifice plate position, tip plate positions and supplies holders.
  • This functional island and other automation equipment such as: automatic picking clone instrument, automatic nucleic acid extraction instrument, etc., are the high-throughput and automatic construction process of protein saturation mutation library involved in the present invention; automatic sorting process of saturation mutation library; automatic inoculation and cultivation ; Automated cell processing, antibacterial activity characterization, and automated mass spectrometry characterization processes provide strong support, and edit the relevant process into an automated program script to realize the corresponding automated experimental process.
  • this example refers to the method of Example 1, carries out corresponding engineering transformation on nisin, and fuses the green fluorescent protein GFP before the nisin guide peptide, It is convenient to observe the expression of the target protein (FIG. 11A), and at the same time, insert the LicP enzyme cleavage site NDVDPE into the nisin leader peptide and core peptide (as shown in FIG. 11B).
  • the Licp enzyme is positioned in the periplasmic space, and Nisin and its corresponding modification enzyme NisinB/C are expressed in the cytoplasm.
  • Nisin When Nisin is modified, switch the temperature to 38°C to lyse E.coli, so that The LicP enzyme contacts the modified nisin and hydrolyzes to release the mature nisin peptide.
  • the antibacterial activity of nisin was verified by the agar antibacterial experiment in Example 1, and the nisin product was also detected by MALDI-TOF MS ( Figure 11C). This experiment proves that the lanthipeptide high-throughput screening platform is suitable for the expression and activity detection of various lanthipeptide antibiotics.

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Abstract

La présente invention concerne un procédé de criblage à haut débit de lantibiotiques. Le procédé comprend la construction d'une banque de mutagénèse par saturation ciblant le site de la lanthiopeptine, l'extraction de plasmides pour transformer des souches d'expression afin d'obtenir des mutants ; la caractérisation de l'activité antibactérienne comprend l'obtention de l'activité antibactérienne de chaque mutant au moyen d'un dosage d'inhibition bactérienne pour réaliser un criblage automatisé à haut débit des activités des lantibiotiques, et la détection de l'échantillon comprend la détection automatisée à haut débit par MALDI-TOF d'un produit cible. La présente invention permet la construction et le criblage automatisés à haut débit d'une banque de mutagénèse par saturation de sites lantibiotiques à l'aide d'une station de travail automatisée.
PCT/CN2022/078528 2022-03-01 2022-03-01 Procédé de criblage à haut débit de lantibiotiques WO2023164788A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101754769A (zh) * 2007-07-18 2010-06-23 诺瓦克塔生物系统有限公司 基于具有抗微生物活性的b型羊毛硫抗生素的用途
US20150050256A1 (en) * 2012-02-07 2015-02-19 The Board Of Trustees Of The University Of Illinois Class I and II Lantibiotics from Geobacillus thermodenitrificans
US20170204400A1 (en) * 2014-09-26 2017-07-20 The Board Of Trustees Of The University Of Illinois Biosynthesis and engineering of lanthipeptides
US20180118791A1 (en) * 2016-10-28 2018-05-03 The Texas A&M University System Modified lantibiotics and methods of making and using the modified lantibiotics
CN112239490A (zh) * 2019-07-16 2021-01-19 武汉臻智生物科技有限公司 羊毛硫肽的筛选方法及无细胞蛋白合成体系和羊毛硫肽

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101754769A (zh) * 2007-07-18 2010-06-23 诺瓦克塔生物系统有限公司 基于具有抗微生物活性的b型羊毛硫抗生素的用途
US20150050256A1 (en) * 2012-02-07 2015-02-19 The Board Of Trustees Of The University Of Illinois Class I and II Lantibiotics from Geobacillus thermodenitrificans
US20170204400A1 (en) * 2014-09-26 2017-07-20 The Board Of Trustees Of The University Of Illinois Biosynthesis and engineering of lanthipeptides
US20180118791A1 (en) * 2016-10-28 2018-05-03 The Texas A&M University System Modified lantibiotics and methods of making and using the modified lantibiotics
CN112239490A (zh) * 2019-07-16 2021-01-19 武汉臻智生物科技有限公司 羊毛硫肽的筛选方法及无细胞蛋白合成体系和羊毛硫肽

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