A scarless gene editing system for Sphingomonas and its application

Abstract

This invention belongs to the field of gene editing technology, specifically relating to a scarless gene editing system for *Sphingomonas* and its application. The scarless gene editing system comprises a first inducing plasmid, a donor plasmid, and a second inducing plasmid. The first inducing plasmid includes the following elements: the EXO gene, the Gam gene, the Beta gene, a tetracycline resistance gene, and replicon 3233. The donor plasmid includes an exogenous DNA fragment for homologous recombination capable of amplifying upstream and downstream homologous arms of the target gene, an FRT fragment, and a chloramphenicol resistance gene. The second inducing plasmid includes the following elements: the FLP gene, a bleomycin resistance gene, and replicon 3233. Based on this scarless gene editing system, this invention knocks out the *Sphingomonas* 1535 gene, achieving an engineered strain capable of producing zeaxanthin, and further knocks out the 516 gene to increase zeaxanthin yield.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to a scarless gene editing system for Sphingomonas and its application. Background Technology

[0002] Zeaxanthin is a yellow-orange carotenoid mainly found in dark green leafy vegetables and egg yolks. Like lutein, zeaxanthin accumulates primarily in the central retina of animals and humans, providing photoprotection against strong light damage and improving retinal activity. Furthermore, multiple clinical trials have shown that combined treatment with lutein and zeaxanthin can further reduce the risk of macular degeneration. Zeaxanthin can also effectively reduce colonic inflammation caused by acetic acid-induced ulcerative colitis by enhancing antioxidant defense mechanisms.

[0003] In recent years, with the rise and development of the synthetic biology industry, various engineered strains have been used for carotenoid production. Sphingosine monocytogenes is a type of Gram-negative, rod-shaped, heterotrophic, aerobic bacterium widely distributed in nature. Currently, Sphingosine monocytogenes is widely used in industrial production for the synthesis of extracellular polysaccharides and has been deemed safe by the U.S. Food and Drug Administration (FDA), receiving approval from both the U.S. and European Unions for use in the food industry. Furthermore, Sphingosine monocytogenes possesses an endogenous carotenoid biosynthetic pathway, capable of synthesizing various carotenoids, including β-carotene, zeaxanthin, astaxanthin, and novoxanthin.

[0004] Previous studies have constructed genetically engineered Sphingomonas strains that produce high levels of zeaxanthin, such as using suicide plasmid-mediated homologous recombination to knock out the target gene. However, problems such as excessively long knockout cycles and low knockout rates still exist. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to provide a Sphingomonas gene editing system and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a scarless gene editing system for Sphingomonas, the gene editing system comprising a first inducing plasmid, a donor plasmid, and a second inducing plasmid; The first inducing plasmid includes the following elements: EXO gene, Gam gene, Beta gene, tetracycline resistance gene, and replicon 3233; the donor plasmid includes an exogenous DNA fragment for homologous recombination that can amplify upstream and downstream homologous arms of the target gene, FRT fragment, and chloramphenicol resistance gene; the second inducing plasmid includes the following elements: FLP gene, bleomycin resistance gene, and replicon 3233.

[0007] In some embodiments, the order of elements in the exogenous DNA fragment used for homologous recombination is: upstream homologous arm of the target gene - FRT site - chloramphenicol resistance gene - FRT site - downstream homologous arm of the target gene.

[0008] In some embodiments, the first inducing plasmid is a pKD46-3233-Tet plasmid carrying EXO, Gam, Beta protein expression genes, tetracycline (Tet) resistance genes, and a fragment of Sphingomonas replicon 3233, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0009] In some embodiments, the donor plasmid is a pACYC-500-FRT-Cm plasmid carrying 500bp homologous arms upstream and downstream of the target gene, an FRT fragment, and a chloramphenicol (Cm) resistance gene, which can amplify exogenous DNA fragments containing 500bp homologous arms upstream and downstream of the target gene, an FRT fragment, and a chloramphenicol (Cm) resistance gene for homologous recombination.

[0010] In some embodiments, the second inducing plasmid is a pKD46-FLP-3233-BleR plasmid carrying the FLP protein expression gene, the bleomycin (BleR) resistance gene, and the sphingosine mononucleosis replicon 3233 fragment, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0011] In some embodiments, constructing the donor plasmid specifically includes: using the Sphingomonas CW1 genome as a template, amplifying a DNA fragment containing upstream and downstream homologous arms of the target gene using designed primers, and inserting the DNA fragment into a first inducing plasmid using homologous recombination to obtain a recombinant plasmid; subsequently, using the pKD4 plasmid as a template, amplifying a linearized fragment (SEQ ID NO: 3) using KD4-Cm-for / rev primers, and using the pACYC184 plasmid as a template, amplifying the Cm resistance gene (SEQ ID NO: 4) using Cm-for / rev primers, and obtaining a plasmid pKD4-Cm containing the Cm resistance gene through homologous recombination of the fragments such as SEQ ID NO: 3 and SEQ ID NO: 4; using the pKD4-Cm plasmid as a template, amplifying an FRT-Cm-FRT fragment using primers designed to contain the FRT fragment, and inserting the FRT-Cm-FRT fragment into the middle of the upstream and downstream homologous arms of the target gene in the recombinant plasmid through homologous recombination to obtain the donor plasmid.

[0012] A second aspect of the present invention provides the application of the above-described traceless gene editing system in editing the genome of Sphingosine Monoclonalella.

[0013] A third aspect of the present invention provides a method for scarless gene editing of Sphingomonas, comprising: Construct a first inducing plasmid, wherein the first inducing plasmid comprises, in sequence, an EXO gene, a Gam gene, a Beta gene, a tetracycline resistance gene and a replicon 3233; Construct a donor plasmid comprising, in sequence, an upstream homologous arm of the target gene, an FRT site, a chloramphenicol resistance gene, an FRT site, and a downstream homologous arm of the target gene; Construct a second inducing plasmid containing, in sequence, an FLP gene, a bleomycin resistance gene, and a replicon 3233; The first inducing plasmid was transferred into competent cells of *Sphingomonas* to obtain the first engineered strain. Using the donor plasmid as a template, an exogenous DNA fragment for homologous recombination was amplified. The exogenous DNA fragment was transferred into competent cells of the first engineered strain and activated in a medium containing chloramphenicol to obtain the second engineered strain. The second inducing plasmid was transferred into competent cells of the second engineered strain to act on the FRT site integrated into the genome, causing the loss of the resistance gene, thus obtaining the third engineered strain, which is the *Sphingomonas* gene-edited mutant strain.

[0014] In some embodiments, the first inducing plasmid is further transformed into competent cells of a third engineered strain and cultured in a medium containing tetracycline to obtain a fourth engineered strain. The fourth engineered strain is then subjected to a second gene editing by further transforming it with an exogenous DNA fragment containing a second target gene and a second inducing plasmid.

[0015] In some embodiments, the nucleotide sequence of the first inducing plasmid is shown in SEQ ID NO: 1, and the nucleotide sequence of the second inducing plasmid is shown in SEQ ID NO: 2.

[0016] In some embodiments, the transfer method is electroconversion.

[0017] In some embodiments, the conditions for the electrical conversion are: 2.5 kV, 25 μF, 200 Ω, 2 mm.

[0018] A fourth aspect of the present invention provides a method for increasing the yield of zeatin in Sphingomonas, comprising performing multi-gene scarless editing of Sphingomonas using the scarless gene editing system described in the present invention.

[0019] In some embodiments, the multi-gene scarless editing specifically involves knocking out genes 1535 and 516 in Sphingomonas CW1.

[0020] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention achieves scarless gene editing by constructing the pKD46-3233-Tet plasmid carrying EXO, Gam, Beta protein expression genes, tetracycline (Tet) resistance genes, and the 3233 fragment of the Sphingomonas replicon. This plasmid is then electroporated into Sphingomonas. Subsequently, the pACYC-500-FRT-Cm plasmid is constructed, carrying 500bp homologous arms upstream and downstream of the target gene, an FRT fragment, and a chloramphenicol (Cm) resistance gene. This amplifies the DNA fragment containing the 500bp homologous arms upstream and downstream of the target gene, the FRT fragment, and the chloramphenicol (Cm) resistance gene, and electroporates it into Sphingomonas to knock out the target gene. Following this, the pKD46-FLP-3233-BleR plasmid is constructed, carrying the FLP protein expression gene, bleomycin (BleR) resistance gene, and the 3233 fragment of the Sphingomonas replicon. The inserted Cm resistance gene is then removed, thus enabling traceless gene editing and allowing for the further knockout of a second gene. Based on this traceless gene editing system, this invention knocks out the 1535 gene of Sphingomonas sphingosine monocytogenes to achieve an engineered strain that can produce zeaxanthin, and further knocks out the 516 gene to increase zeaxanthin yield. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the pKD46-3233-Tet plasmid in an embodiment of the present invention; Figure 2 This is a schematic diagram of the pACYC184-1535-FRT-Cm plasmid in an embodiment of the present invention; Figure 3 This is a schematic diagram of the pACYC184-516-FRT-Cm plasmid in an embodiment of the present invention; Figure 4 This is a schematic diagram of the pKD46-FLP-3233-BleR plasmid in an embodiment of the present invention; Figure 5 These are the genomic PCR detection results of WT strain, Δ1535 strain, and Δ1535Δ516 strain in the embodiments of the present invention; Figure 6 The growth curves of WT strain, Δ1535 strain, and Δ1535Δ516 strain in the embodiments of the present invention are shown. Figure 7 This is a bar chart comparing the maize xanthine yields of WT strain, Δ1535 strain, and Δ1535Δ516 strain in the embodiments of the present invention. Figure 8 The HPLC detection results of fermentation products of WT strain, Δ1535 strain, and Δ1535Δ516 strain in the embodiments of the present invention are shown. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0024] In the following examples, the sphingosomalidobacterium CW1 ( Sphingomonas sp. CW1 was isolated and preserved in our laboratory at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252998, with a preservation date of December 24, 2025. The *E. coli* Top10 competent cells were purchased from Shanghai Bioengineering Co., Ltd. The plasmids pKD46 (GenBank: AY048746.1), pKD4 (GenBank: AY048743.1), pCP20 (GenBank: GM619722.1), and pACYC184 (GenBank: X06403.1) were donated by Researcher Xie Haixia from the Institute of Hydrobiology, Chinese Academy of Sciences.

[0025] In the following examples, the yield of zeaxanthin in zeaxanthin was detected by high performance liquid chromatography (HPLC) under the following conditions: The HPLC system used was a Waters 1525 HPLC system; the column was a 4.6 × 250 mm, 5 μm ThermoScientific C18 reversed-phase column; the detector was a DVD detector; the detector wavelength was 475 nm; the mobile phase was methanol:acetonitrile:dichloromethane = 35:50:15; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the full wavelength scan was 190-800 nm; the isocratic elution program was: the column was equilibrated with 100% mobile phase for 30 min before injection; a total of 30 min of signal acquisition was performed.

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0027] Example 1: Construction of pKD46-3233-Tet plasmid 1. Based on the pKD46 plasmid, linearized fragment A was amplified using pKD46-for / pKD46-rev primers. Based on the pACYC184 plasmid, linearized fragment B was amplified using Tet-for / Tet-rev primers. The linearized fragment amplification system consisted of: 1 μL template, 1 μL for primer, 1 μL rev primer, 25 μL 2×PrimeSTAR Max DNA Polmerase (Takara, catalog number RO45A), and ddH2O to a final volume of 50 μL.

[0028] The linearized fragment amplification reaction was performed using tidal PCR with the following conditions: 98℃ for 10 min; 20 cycles × (98℃ for 10 s; 60-50℃ for 5 s; 72℃ for 5 s); 10 cycles × (98℃ for 10 s; 50℃ for 5 s; 72℃ for 5 s); 72℃ for 10 min; 4℃ for 10 min. For the first 20 cycles, the annealing temperature was decreased by 0.5℃ per cycle; the annealing time was adjusted according to the amplified DNA fragment, requiring 5 s per 1000 bp.

[0029] 2. The linearized fragment obtained after gel recovery and purification was used for homologous recombination to construct the pKD46-Tet plasmid; The homologous recombination reaction system consisted of fragment A, fragment B, 10 μL of 2X MultiF Seamless Assembly Mix (ABclonal, catalog number RK21020), and ddH2O to a final volume of 20 μL. The amounts of fragment A and fragment B added were adjusted according to their sizes, following their respective instructions. The homologous recombination reaction conditions were: 50℃ for 30 min; 4℃ for 10 min.

[0030] 3. Subsequently, based on the pKD46-Tet plasmid, linearized fragment 1 was amplified using 46T-for / 46T-rev primers. Using a synthetic plasmid (Shanghai Sangon Biotech) containing the nucleotide sequence of replicon 3233 as a template, linearized fragment 2 was amplified using rio-for / rio-rev primers. Through homologous recombination, the pKD46-3233-Tet plasmid was constructed, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0031] Table 1 Primers required for constructing pKD46-3233-Tet plasmid

[0032] Example 2: Construction of pACYC-1535-FRT-Cm and pACYC-516-FRT-Cm plasmids Construction of pACYC-1535-FRT-Cm plasmid: Using *Sphingomonas* genome as a template, a DNA fragment containing upstream and downstream 500bp homologous arms was amplified using primers 1535-U-for / 1535-D-rev. Using pKD46-3233-Tet plasmid as a template, a linearized fragment was amplified using primers 184-1535-for / 184-1535-rev. Homologous recombination was then used to construct the pACYC-1535 plasmid. Subsequently, using pKD4 plasmid as a template, a linearized fragment (as shown in SEQ ID NO: 3) was amplified using primers KD4-Cm-for / KD4-Cm-rev. Using pACYC184 plasmid as a template, the Cm resistance gene (as shown in SEQ ID NO: 3) was amplified using primers Cm-for / Cm-rev. (As shown in NO: 4) Homologous recombination was used to construct the pKD4-Cm plasmid. Using the pKD4-Cm plasmid as a template, the linearized fragment was amplified using FRT-1535-for / FRT-1535-rev. Using the pACYC-1535 plasmid as a template, the linearized fragment was amplified using 1535-D-for / 1535-U-rev primers. Homologous recombination was then used to construct the pACYC-1535-FRT-Cm plasmid. The required reaction system and reaction conditions were the same as in Example 1.

[0033] Construction of pACYC-516-FRT-Cm plasmid: Using the *Sphingomonas* genome as a template, a DNA fragment containing upstream and downstream 500 bp homologous arms was amplified using primers 516-U-for / 516-D-rev. Using pKD46-3233-Tet plasmid as a template, the linearized fragment was amplified using primers 184-516-for / 184-516-rev. Homologous recombination was then used to construct the pACYC-516 plasmid. Subsequently, using pKD4 plasmid as a template, KD4-Cm-for / KD4-Cm-re... Using primer v, the linearized fragment was amplified. Using pACYC184 plasmid as a template, the Cm resistance gene was amplified via Cm-for / Cm-rev. Homologous recombination was then used to construct the pKD4-Cm plasmid. Using pKD4-Cm plasmid as a template, the linearized fragment was amplified via FRT-516-for / FRT-516-rev. Using pACYC-516 plasmid as a template, the linearized fragment was amplified via 516-D-for / 516-U primers. Homologous recombination was then used to construct the pACYC-516-FRT-Cm plasmid. The required reaction system and conditions were the same as in Example 1.

[0034] Table 2 Primers required for constructing pACYC-1535-FRT-Cm and pACYC-516-FRT-Cm plasmids

[0035] Example 3: Construction of pKD46-FLP-3233-BleR plasmid Using pKD46-3233-Tet plasmid as a template, the linearized fragment was amplified using primers 46-FLP-for / 46-FLP-rev. Using pCP20 plasmid as a template, the FLP expression gene was amplified using primers FLP-46-for / FLP-46-rev. Homologous recombination was then used to construct the pKD46-3233-FLP plasmid. Subsequently, using pKD46-3233-FLP plasmid as a template, the linearized fragment was amplified using primers 46-BleR-for / 46-BleR-rev. Using pPICZaA plasmid as a template, the Ble resistance gene was amplified using BleR-for / BleR-rev. Homologous recombination was then used to construct the pKD46-FLP-3233-BleR plasmid, the nucleotide sequence of which is shown in SEQ ID NO: 2. The required reaction system and conditions were the same as in Example 1.

[0036] Table 3 Primers required for constructing pKD46-FLP-3233-BleR plasmid

[0037] Example 4: Sphingosine Monoclonal gene knockout 1. Take a streak plate of Sphingomonas. After 48 hours, pick a single colony and transfer it to 5 ml of LB medium. After 48 hours, transfer it to 50 ml of LB medium. After about 30 hours (logarithmic growth phase), centrifuge and remove the supernatant. Add 25 ml of ice-cold ddH2O, incubate on ice for 10 minutes, then centrifuge at 4°C and 5000 rpm for 10 minutes, and remove the supernatant. Add another 25 ml of ice-cold ddH2O, incubate on ice for 10 minutes, then centrifuge at 4°C and 5000 rpm for 10 minutes, and remove the supernatant. Add 25 ml of ice-cold 10% glycerol solution, incubate on ice for 10 minutes, then centrifuge at 4°C and 5000 rpm for 10 minutes, and remove the supernatant. Add 200 μl of ice-cold 10% glycerol solution for electroporation.

[0038] 2. Take 200 μL of wild-type Sphingomonas sphingosine mononitrate competent cells, add 500 ng of pKD46-3233-Tet plasmid, incubate on ice for 10 min, and then perform electroporation under the following conditions: 2.5 kV, 25 μF, 200 Ω, 2 mm. Then add 1 mL of LB+Tet medium and activate at 28℃ for 3-4 h. Spread the medium on LB+Tet plates, pick single clones, and detect them using the pKD46-3233-Tet plasmid and primers Gam-for / Beta-rev (Table 5).

[0039] 3. Prepare competent cells from WT / pKD46-3233-Tet. Using pACYC-1535-FRT-Cm plasmid as a template, amplify the linearized fragment for homologous recombination using 1535-U-for / 1535-D-rev (Table 2) as primers. Add 10 μL of the linearized fragment to 200 μL of Sphingomonas competent cells and electroporate (see step 2 for electroporation conditions). Then add 1 mL of LB+Cm34 medium and activate at 28℃ for 3-4 h. Spread the culture onto LB+Cm34 plates. After the culture has grown, pick single colonies and transfer them to LB+Cm34 medium. After growth, extract the genome and perform PCR detection. The detection conditions are as described in step 1 of Implementation Case 1. The detection primers are 1535-C-for / 1535-C-rev in Table 4. Construct the Sph / Δ1535::Cm / pKD46-3233-Tet strain.

[0040] 3. Prepare competent cells using Sph / Δ1535::Cm / pKD46-3233-Tet. Add 3-4 μL of pKD46-FLP-3233-BleR to 200 μL of competent cells, electroporate, and plate on LB+Ble50 plates. After single colonies grow, pick single colonies and streak them onto LB+Ble50 and LB+Cm34 plates. Select single colonies that grow on LB+Ble50 plates but not on LB+Cm34 plates and transfer them to LB+Ble50 medium. After growth, extract the genome and perform PCR detection. The detection conditions are as described in step 1 of Example 1. The detection primers are 1535-C-for / 1535-C-rev from Table 4. The 1535 gene knockout Sph / Δ1535 / pKD46-FLP-3233-BleR strain was obtained. The detection results are... Figure 5 Comparison of PCR results between wild type and Δ1535.

[0041] Table 4. Primers for detecting strains with gene deletions of 1535 and 516.

[0042] Example 5: Removal of resistance gene and secondary gene knockout in Sphingomonas sphingosine mononitrate strains 1. Prepare competent cells using Sph / Δ1535 / pKD46-FLP-3233-BleR from Example 4. Add 3-4 μL of pKD46-3233-Tet to 200 μL of competent cells, electroporate, and plate on LB+Tet50 plates. After single colonies grow, pick single colonies and streak them on LB+Tet50 and LB+Ble50 plates. Select single colonies that grow on LB+Tet50 plates but not on LB+Ble50 plates and transfer them to LB+Tet50 medium. After growth, perform colony PCR detection on the bacterial culture and select single colonies that can only detect pKD46-3233-Tet.

[0043] 2. Using pACYC-516-FRT-Cm plasmid as a template, and with 516-U-for / 516-D-rev (Table 2) as primers, amplify the linearized fragment for homologous recombination; add 10 μL of the linearized fragment to 200 μL of Sphingosine monocytogenes competent cells, electroporate (electroplation conditions: see step 2 of Implementation Case 4), then add 1 mL of LB+Cm34 medium and activate at 28℃ for 3-4 h; then plate on LB+Cm34 plates, and after the plates have grown, pick single colonies and transfer them to LB+Cm34 medium. After growth, extract the genome and perform PCR detection. The detection conditions are as described in step 1 of Implementation Case 1, and the detection primers are 516-C-for / 516-C-rev in Table 4.

[0044] 3. Prepare competent cells using Sph / Δ1535Δ516::Cm / pKD46-3233-Tet. Add 3-4 μL of pKD46-FLP-3233-BleR to 200 μL of competent cells, electroporate, and plate on LB+Ble50 plates. After single colonies grow, pick single colonies and streak them on LB+Ble50 and LB+Cm34 plates. Select single colonies that grow on LB+Ble50 plates but not on LB+Cm34 plates and transfer them to LB+Ble50 medium. After growth, extract the genome and perform PCR detection. The detection conditions are as described in step 1 of Implementation Case 1. The detection primers are 1535-C-for / 1535-C-rev in Table 4. The *Sphomonas* strain Sph / Δ1535Δ516 / pKD46-FLP-3233-BleR, with both genes 1535 and 516 knocked out, was obtained. The detection results are as follows: Figure 5 As shown.

[0045] Table 5 Primers required for detection using pKD46-3233-Tet plasmid and pKD46-FLP-3233-BleR plasmid.

[0046] Example 6: Growth Curve Measurement Streaking LB agar plates containing *Sphingomonas WT*, Δ1535, and Δ1535Δ516 were taken and incubated at 28°C for 48 h. Single colonies were then picked and transferred to 5 ml of LB agar, and incubated at 28°C with a shaker at 180 rpm for 48 h. OD600 was then measured. The culture was then transferred to 50 ml of LB agar at the same inoculum size and incubated at 28°C with a shaker at 180 rpm for 120 h. Every 24 h, 2 ml of bacterial culture was collected, centrifuged at 5000 rpm for 10 min, and the precipitate was collected. All samples were then lyophilized and weighed. The growth curves of the three *Sphingomonas WT*, Δ1535, and Δ1535Δ516 are shown below. Figure 6 As shown.

[0047] Example 7 Fermentation Production of Zeaxanthin 1. Take Sphingomonas WT, Δ1535, Δ1535Δ516 and streak them on LB agar plates. After incubation at 28°C for 48 h, pick single colonies and transfer them to 5 mL of LB agar. Incubate at 28°C and shake at 180 rpm for 48 h. Then measure the OD600. Transfer the same inoculum to 50 mL of LB agar and incubate at 28°C and shake at 180 rpm for 120 h. Then centrifuge the bacterial suspension at 5000 rpm for 10 min to collect the precipitate. Add 25 mL of purified water to suspend the bacterial cells. Centrifuge at 5000 rpm for 10 min to collect the precipitate. Freeze the sample at -80°C for 12 h overnight. Take it out and freeze it in a freeze dryer for 12 h. Weigh the dry weight of the bacterial cells.

[0048] 2. Take the dried bacterial cells and grind them using a high-throughput tissue homogenizer. Then add acetone to extract carotenoids. Repeat the extraction until the broken tissue is white. Filter the extract through a 0.22μm organic filter membrane and then perform HPLP detection. Figure 7 For comparison of maize xanthine yield, Figure 8 The fermentation products of three strains of Sphingosine Monoclonalella (WT, Δ1535, and Δ1535Δ516) were detected.

[0049] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A scarless gene editing system for Sphingomonas, characterized in that, The scarless gene editing system comprises a first inducing plasmid, a donor plasmid, and a second inducing plasmid. The first inducing plasmid includes the following elements: EXO gene, Gam gene, Beta gene, tetracycline resistance gene, and replicon 3233; The donor plasmid includes an exogenous DNA fragment for homologous recombination that can amplify upstream and downstream homologous arms of the target gene, an FRT fragment, and a chloramphenicol resistance gene. The second inducing plasmid includes the following elements: the FLP gene, the bleomycin resistance gene, and replicon 3233.

2. The scarless gene editing system as described in claim 1, characterized in that, The nucleotide sequence of the replicon 3223 is shown in SEQ ID NO:

1.

3. The scarless gene editing system as described in claim 1, characterized in that, The order of elements in the exogenous DNA fragment used for homologous recombination is: upstream homologous arm of the target gene - FRT site - chloramphenicol resistance gene - FRT site - downstream homologous arm of the target gene.

4. The use of the scarless gene editing system according to any one of claims 1-3 in editing the genome of Sphingosine Monoclonalella.

5. A method for scarless gene editing of Sphingomonas, characterized in that, include: Construct a first inducing plasmid, wherein the first inducing plasmid comprises, in sequence, an EXO gene, a Gam gene, a Beta gene, a tetracycline resistance gene and a replicon 3233; Construct a donor plasmid comprising, in sequence, an upstream homologous arm of the target gene, an FRT site, a chloramphenicol resistance gene, an FRT site, and a downstream homologous arm of the target gene; Construct a second inducing plasmid containing, in sequence, an FLP gene, a bleomycin resistance gene, and a replicon 3233; The first inducing plasmid was transferred into competent cells of *Sphingomonas* to obtain the first engineered strain. Using the donor plasmid as a template, an exogenous DNA fragment for homologous recombination was amplified. The exogenous DNA fragment was transferred into competent cells of the first engineered strain and activated in a medium containing chloramphenicol to obtain the second engineered strain. The second inducing plasmid was transferred into competent cells of the second engineered strain to act on the FRT site integrated into the genome, causing the loss of the resistance gene, thus obtaining the third engineered strain, which is the *Sphingomonas* gene-edited mutant strain.

6. The method as described in claim 5, characterized in that, The first inducing plasmid was further transformed into competent cells of the third engineered strain and cultured in a medium containing tetracycline to obtain the fourth engineered strain. The fourth engineered strain was then subjected to a second gene editing by further transforming it with an exogenous DNA fragment containing the second target gene and the second inducing plasmid.

7. The method as described in claim 5, characterized in that, The nucleotide sequence of the first inducing plasmid is shown in SEQ ID NO: 1, and the nucleotide sequence of the second inducing plasmid is shown in SEQ ID NO:

2.

8. The method as described in claim 5, characterized in that, The transfer method is electroconversion.

9. A method for increasing zeatin yield in Sphingomonas sphingosine monocytogenes, characterized in that, Multiple genes of Sphingosine monocytogenes were edited without scarring using the gene editing system described in any one of claims 1-3.

10. The method as described in claim 9, characterized in that, The aforementioned multi-gene scarless editing specifically involves knocking out genes 1535 and 516 in Sphingosine monocytogenes.

Images