Serratia marcescens engineering bacterium for high yield of prodigiosin as well as construction method and application of serratia marcescens engineering bacterium

Abstract

The invention discloses serratia marcescens engineering bacteria with high yield of prodigiosin as well as a construction method and application of the serratia marcescens engineering bacteria. Through a gene engineering technology, a dapB gene of serratia marcescens is knocked out by utilizing a Red homologous recombination system, and a dapB defect type engineering strain is constructed. After the strain is subjected to shake-flask culture in a fermentation culture medium for 60 hours, the yield of prodigiosin reaches 0.91 g / L and is increased by 25.5% compared with that of a wild strain. The construction method of the engineering bacteria is simple and convenient, genetic stability is achieved, and a new way is provided for efficient microbial production of prodigiosin.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering and strain modification technology, specifically to an engineered strain of Serratia marcescens that produces high levels of erythromycin, its construction method, and its application. Background Technology

[0002] Prodigiosin is a natural tripyrrole red pigment belonging to the quinone class of compounds, produced by various bacteria such as Serratia marcescens and actinomycetes. This pigment is biodegradable and environmentally friendly, showing broad application potential in the pharmaceutical, agricultural, industrial, and environmental fields.

[0003] In the pharmaceutical field, lindane exhibits a variety of biological activities, including anticancer, immunosuppressive, and antimalarial effects; in the agricultural and environmental fields, it has broad-spectrum antibacterial, antifungal, and algae-inhibiting capabilities; and in industry, it is used as a natural red pigment in dye preparation.

[0004] Currently, the main methods for synthesizing styraxin include chemical synthesis and microbial synthesis. Chemical synthesis is costly and inefficient, making it difficult to achieve large-scale production; while microbial synthesis, represented by Serratia marcescens, has advantages such as mild conditions, environmental friendliness, and ease of industrialization, making it the main production method.

[0005] The biosynthesis of styraxin is mainly composed of pig Gene clusters (including) pigA-N Controlled by genes such as β-carotene, the precursors MBC and MAP are synthesized through two branching pathways, ultimately condensing under PigC catalysis to form squalene. The MBC synthesis pathway has a heavy metabolic burden and is one of the key factors limiting yield.

[0006] Although wild-type Serratia marcescens can produce styraxin, its yield is still insufficient to meet industrial demand. Therefore, modifying strains through genetic engineering to increase styraxin production has significant research value and application prospects. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a high-yield Serratia marcescens engineered strain of sclerotinib, its construction method, and its applications, achieved by knocking out... dapB Genes are used to optimize the metabolic pathways of strains, thereby improving the efficiency of styraxin synthesis.

[0008] This invention provides a high-yield strain of *Serratia marcescens* producing sclerotin, its construction method, and its application, employing the following technical solution: In a first aspect, the present invention provides an engineered strain of *Serratia marcescens* that produces high levels of styraxanthin, wherein the engineered strain of *Serratia marcescens* encodes dihydropyridine dicarboxylic acid reductase. dapB The gene was knocked out.

[0009] Preferably, the dapB The gene was replaced by a kanamycin resistance gene through homologous recombination.

[0010] A second aspect of the present invention provides a method for constructing the above-mentioned engineered Serratia marcescens bacteria, comprising the following steps: Step 1): Using Serratia marcescens genomic DNA as a template, PCR amplification... dapB The upstream and downstream homologous arms of the gene; containing the kanamycin resistance gene ( KanR Using the plasmid as a template, PCR amplification was performed. KanR Fragment; Linearization of the suicide plasmid pDM4 by reverse PCR; Step 2): Using seamless cloning technology, the upstream homologous arm, kanamycin resistance gene fragment, and downstream homologous arm obtained in Step 1) are ligated with linearized pDM4 to obtain the recombinant suicide plasmid pDM4- ΔdapB ; Step 3): The recombinant suicide plasmid pDM4- obtained in Step 2) ΔdapB Wild-type Serratia marcescens was introduced via conjugation transfer. Step 4): Obtain the results through a first-round exchange filter and a second-round exchange filter. dapB After knocking out the bacteria, colony PCR verification and sequencing confirmed that the engineered Serratia marcescens strain was obtained.

[0011] A third aspect of the present invention provides an application of the above-mentioned engineered Serratia marcescens strain in increasing the yield of serotonin.

[0012] Colonies were picked and inoculated into LB medium for seed culture. A 5% inoculum was then added to the fermentation medium (fructose 16.29 g / L, peptone 11.76 g / L, Tween 80 2.64 g / L, initial pH 6.0), and cultured at 30℃ and 160 rpm for 60 h. The results showed that the constructed engineered *Serratia marcescens* strain produced 25.5% more styraxanthin than the wild-type control strain.

[0013] This invention is the first to knock out Serratia marcescens. dapB The gene significantly increased the yield of styraxin, providing a new technical solution for the efficient production of styraxin by microbial methods.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to knock out *Serratia marcescens* based on homologous recombination of a suicide plasmid. dapB The genetically engineered Serratia marcescens strain obtained by constructing the gene significantly increased the production of styraxone in Serratia marcescens, with a 25.5% increase in styraxone production compared to the wild-type control strain.

[0015] 2. This invention introduces the kanamycin gene into homologous recombination, which significantly improves the efficiency of recombination screening and provides a new recombination screening method.

[0016] 3. The increased production of lecithin in Serratia marcescens has high application value in the fields of anti-cancer, antibacterial, and biological staining. Attached Figure Description

[0017] Figure 1 The suicide knockout plasmid pDM4- of this invention ΔdapB A schematic diagram of the construction process; Figure 2 In Embodiment 1 of the present invention, the upstream homologous arm, KanR PCR validation results of the fragment and downstream homologous arm. Lane M is the DNA Marker, and lanes 1, 2, and 3 are the upstream homologous arm, downstream homologous arm, and downstream homologous arm, respectively. KanR Amplification products of fragments and downstream homologous arms; Figure 3 The image shows the results of reverse PCR linearization of plasmid pDM4 in Example 1 of this invention. Lane M in the electrophoresis diagram is the DNA Marker, and lane 1 is the linearized plasmid pDM4. Figure 4 This is a PCR verification result diagram of the single-exchange recombinant strain in Example 2 of the present invention. Lane M in the electrophoresis diagram is the DNA Marker, lane 1 is the wild-type control, and lanes 2-5 are the single-exchange verified strains. Figure 5 This is a PCR verification result diagram of the double-exchange recombinant strain in Example 3 of the present invention. Lane M in the electrophoresis diagram is the DNA Marker, lane 1 is the wild-type control, and lanes 2-5 are the strains that were successfully verified by double exchange. Figure 6 This is a comparison chart of the strychnine production of engineered and wild-type Serratia marcescens strains in Example 4 of the present invention. Detailed Implementation

[0018] To make the present invention more apparent and understandable, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] This invention provides an engineered strain of *Serratia marcescens* that produces high levels of styraxanthin, along with its construction method and applications. Through genetic engineering technology, the Red homologous recombination system is used to knock out *Serratia marcescens*. dapB Genes, construction dapB A defective engineered strain was developed. After 60 hours of shake-flask culture in fermentation medium, this strain achieved a yield of 0.91 g / L of styracil, which is 25.5% higher than that of the wild-type strain. The engineered strain described in this invention is simple to construct and genetically stable, providing a new approach for the efficient microbial production of styracil.

[0020] Unless otherwise specified, the molecular biology experimental methods used in the following examples were performed under standard conditions, referring to "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor).

[0021] Biological material: Serratia marcescens ( Serratia marcescens The strain SM was purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 21537; the suicide plasmid pDM4 was purchased from Miaoling Biotechnology Co., Ltd.; the plasmid pET28a was a laboratory-preserved plasmid. KanR Resistance gene template; Escherichia coli ( E. coli S17-1 λpir was used for plasmid construction and conjugation transfer experiments. Among them, the engineered *Serratia marcescens* strain producing high levels of erythromycin was derived from the wild-type *Serratia marcescens* SM strain by knockout. dapB Gene, dapB The gene nucleotide sequence is shown in SEQ ID NO.1: .

[0022] The construction method is as follows: amplification was performed using PCR. dapB upstream homology arm, downstream homology arm and KanR The resistance gene fragments were ligated in one step using seamless cloning technology to the suicide plasmid pDM4, which had been linearized by reverse PCR. The ligation product was then transformed. E. coliS17-1 competent cells were plated on Kan and Cm antibiotic-protected plates for screening positive transformants, and the gene was obtained after sequencing verification. dapB Knockout plasmid pDM4- ΔdapB pDM4- ΔdapB The plasmid was introduced into wild-type Serratia marcescens SM via conjugation transfer, and after two homologous recombination screenings, a successful knockout was obtained. dapB The engineered strain of the gene was named *Serratia marcescens*. ΔdapB -SM.

[0023] Example 1: E. coli S17-1 / pDM4- ΔdapB Construction of recombinant suicide plasmids (e.g.) Figure 1 (As shown) 1.1 dapB upstream homologous arm of gene, dapB Downstream homologous arms of genes KanR PCR amplification of fragments and linearized pDM4 plasmid fragments Based on the Serratia marcescens SM genome sequence ( dapB Primers were designed for the gene region, and PCR amplification was performed using SM genomic DNA as a template, as shown in SEQ ID NO.2 and SEQ ID NO.3. dapB The upstream homologous arm of the gene (approximately 500 bp) was amplified by PCR using primers shown in SEQ ID NO.4 and SEQ ID NO.5. dapB The downstream homologous arm of the gene (approximately 500 bp); using the laboratory-preserved plasmid pET28a as a template, PCR amplification was performed using primers shown in SEQ ID NO. 6 and SEQ ID NO. 7. KanR The resistance gene fragment (927 bp); using the suicide plasmid pDM4 as a template, a linearized vector fragment (approximately 7000 bp) was obtained by reverse PCR amplification using primers shown in SEQ ID NO. 8 and SEQ ID NO. 9. All PCR products were verified by agarose gel electrophoresis (e.g., ...). Figure 2 and Figure 3 (As shown), the DNA was recovered using a DNA purification kit.

[0024] The primer sequences are as follows: Upstream homologous arm-F (SEQ ID NO.2): 5'-AAGGGCCCCACTAGTGACGCCGTGCTGCTCGATGAAGTAC-3'; Upstream homologous arm-R (SEQ ID NO.3): 5'-AGATCAAAGGATCTTCGCTTGCTCTCTTCTTGATTTGACG-3'; Downstream homologous arm-F (SEQ ID NO.4): 5'-TGCTCGATGAGTTTTTCTAATTGATTTGCATACCATCGTGATG-3'; Downstream homologous arm-R (SEQ ID NO.5): 5'-TACCGTCGACCCTCGAGTACCAAGCGTCCAACTGCCTTG-3'; KanR -F (SEQ ID NO.6): 5'-AAGAAGAGAGCAAGCGAAGATCCTTTGATCTTTTCTACGG-3'; KanR -R (SEQ ID NO.7): 5'-CACGATGGTATGCAAATCAATTAGAAAAACTCATCGAGCATCAAA; Reverse-F (SEQ ID NO.8): 5'-GCGTCACTAGTGGGGCCCTT-3'; Reverse-R (SEQ ID NO. 9): 5'-GTACTCGAGGGTCGACGGTA-3'.

[0025] The PCR system (50 μL) is as follows:

[0026] PCR reaction program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 35 cycles; final extension at 72℃ for 5 min.

[0027] 1.2 Connection Reaction Purified dapB Upstream homologous arm, dapB Downstream homologous arm, KanR The fragments and linearized pDM4 vector fragments were mixed in a molar ratio of 2:2:2:1; using Novizan CloneExpress. ® The cloning reaction was performed using the MultiS One Step Cloning Kit; the reaction system (10 μL) is as follows:

[0028] The reaction conditions are as follows:

[0029] 1.3 Transformation, screening, and identification of ligation products Add all 10 μL of the ligation product to a 100 μL volume. E. coli S17-1 chemocompetent cells were incubated on ice for 30 min; after heat shock at 42℃ for 45 s, they were immediately incubated on ice for 2 min; 900 μL of antibiotic-free LB liquid medium was added, and the cells were revived and cultured at 37℃ and 225 rpm for 1 h; after centrifugation at 5000 rpm for 5 min, 900 μL of supernatant was discarded, and the cells were resuspended in the remaining medium and spread on LB solid plates containing kanamycin (Kan, 50 μg / mL) and chloramphenicol (Cm, 25 μg / mL), and incubated upside down at 37℃ for 12-16 h.

[0030] 1.4 Identification of Recombinant Products Single colonies grown on the plates were picked and inoculated into LB liquid medium containing Kan and Cm antibiotics, and cultured overnight at 37°C. Plasmids were extracted, and PCR verification was performed using vector primers. The samples were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced strain was the desired recombinant bacterium and named... E. coli S17-1 / pDM4- ΔdapB Store at -80℃.

[0031] Example 2: Screening and Identification of Single-Exchange Recombinant Strains Recombinant donor bacteria E. coli S17-1 / pDM4- ΔdapB Streaking was performed on wild-type Serratia marcescens SM, the recipient bacterium, to activate the bacteria. Single colonies were picked and inoculated into LB liquid medium (donor bacteria Kan+Cm, recipient bacteria without antibiotics) and cultured overnight at 37°C (donor bacteria) or 30°C (recipient bacteria) and 150 rpm.

[0032] The bonding and transfer steps are as follows: (1) Take 200 μL of the bacterial culture of wild-type SM strain and 400 μL of the recombinant strain. E. coli S17-1 / pDM4- Δ dapB The bacterial cultures were inoculated into antibiotic-free LB liquid medium and incubated at 37°C. E. coli S17-1 / pDM4- ΔdapB Incubate in a shaker at 30℃ (SM) for 3-4 hours (bacterial solution OD600≈0.6). (2) Take 300 μL of wild-type SM strain bacterial culture and 900 μL of recombinant strain. E. coli S17-1 / pDM4- Δ dapBMix the bacterial culture into a new centrifuge tube, centrifuge at 10,000 rpm for 5 min, discard the supernatant, add 50 μL of LB liquid medium to the centrifuge tube, and mix thoroughly. (3) Drop 50 μL of the mixed bacterial culture vertically onto LB solid medium, incubate the plate upright at 37°C for 4 h, then incubate overnight at 30°C, and then add the recombinant suicide plasmid pDM4- ΔdapB Introduce wild-type Serratia marcescens; (4) Scrape the bacterial growth on the plate with a sterile pipette tip and resuspend it in 600 μL of LB medium; take 100 μL of bacterial solution and spread it on a triple antibody LB solid plate containing Kan, Cm and tetracycline (Tet, 30 μg / mL), and incubate it upside down at 30℃ for 24-48 h; (5) Pick a single colony from the plate and perform colony PCR verification; the strain that tests positive by PCR is the single-exchange strain that has undergone one homologous recombination (e.g. Figure 4 (As shown), the single-exchange PCR verification primer sequences are: Single exchange-F (SEQ ID NO.10): 5'-GAAAACGGGGGAATATCATAATTT-3'; Single exchange-R (SEQ ID NO.11): 5'-TCGCGGCCTAGAGCAA-3'.

[0033] Example 3: Screening and Identification of Recombinant Strains with Secondary Exchange (1) Passage the single clone strain to promote its secondary exchange. Passage 4 times with antibiotic-free LB and 2 times with sucrose (5%) LB. Then streak the bacterial solution onto 8% sucrose LB plate. (2) The bacteria on the sucrose plate were spotted onto LB solid plates containing Kan (50 μg / mL) and Cm (50 μg / mL) respectively, and incubated overnight; (3) Select single clones that grow on Kan plates but die on Cm plates, and perform colony PCR identification using primers as shown in SEQ ID NO.12 and SEQ ID NO.13 (e.g. Figure 5 (As shown), and send the PCR product for sequencing verification; preserve the correct strain and name it. ΔdapB -SM; The primer sequences for the secondary exchange verification PCR are as follows: Double exchange-F (SEQ ID NO.12): 5'-CTGCGGCATCAGCCTG-3'; Double exchange-R (SEQ ID NO.13): 5'-TCGCGGCCTAGAGCAA-3'.

[0034] Example 4: ΔdapB-SM production verification (1) Wild-type Serratia marcescens SM and engineered bacteria ΔdapB - SM were streaked onto antibiotic-free LB solid plates and incubated overnight at 30°C; (2) Pick a single colony and inoculate it into 4 mL of LB liquid medium. Incubate overnight at 30°C and 160 rpm to obtain the first-stage seed culture. (3) Take 200 μL of primary seed culture and transfer it to 4 mL of fresh LB medium. Incubate at 30 °C and 160 rpm for 8 h to obtain secondary seed culture. (4) Inoculate the secondary seed culture into a 500 mL shake flask containing 80 mL of fermentation medium at an inoculation rate of 1%; (5) Three parallel groups were set up for each group and fermented under shaking conditions of 30℃ and 160rpm for 60h.

[0035] Yield determination: After fermentation, the cell bodies were collected by centrifugation; squalene was extracted using acidic ethanol (4% 1M HCl ethanol solution); the absorbance of the extract was measured at 535 nm using a UV-Vis spectrophotometer. The yield of squalene was calculated using the following formula: Y = 1.1936X - 0.001, where Y represents the absorbance of the fermentation broth after acidic ethanol extraction at A... 535 The absorbance value measured at the point, X represents the amount of erythromycin produced by the strain, where 1 unit equals 10 mg / L.

[0036] like Figure 6 As shown, engineered Serratia marcescens bacteria ΔdapB The styrax-rich SM yield was 0.91 g / L, a 25.5% increase compared to the wild-type SM (0.725 g / L). Data are expressed as mean ± standard deviation. A t-test analysis showed the difference to be statistically significant. p <0.05).

[0037] It should be noted that those skilled in the art can make various improvements and additions without departing from the scope of this invention, and these improvements and additions should also be considered within the scope of protection of this invention. Any modifications, alterations, and variations made by those skilled in the art without departing from the spirit and scope of this invention, based on the disclosed technical content, are equivalent embodiments of this invention; furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of this invention still fall within the scope of the technical solution of this invention.

Claims

1. A high-yield Serratia marcescens engineered strain of *Serratia marcescens*, characterized in that, The engineered Serratia marcescens strain encodes dihydropyridine dicarboxylic acid reductase. dapB The gene was knocked out.

2. The engineered Serratia marcescens strain according to claim 1, characterized in that, The dapB The gene was replaced by a kanamycin resistance gene through homologous recombination.

3. A method for constructing an engineered Serratia marcescens strain as described in claim 1, characterized in that, Includes the following steps: Step 1): Using Serratia marcescens genomic DNA as a template, PCR amplification... dapB Upstream and downstream homologous arms of the gene; using a plasmid containing the kanamycin resistance gene as a template, amplify the kanamycin resistance gene fragment by PCR; linearize the suicide plasmid pDM4 by inverse PCR; Step 2): Using seamless cloning technology, the upstream homologous arm, kanamycin resistance gene fragment, and downstream homologous arm obtained in Step 1) are ligated with linearized pDM4 to obtain the recombinant suicide plasmid pDM4- ΔdapB ; Step 3): The recombinant suicide plasmid pDM4- obtained in Step 2) ΔdapB Wild-type Serratia marcescens was introduced via conjugation transfer. Step 4): Obtain the results through a first-stage and a second-stage exchange screening. dapB After knocking out the bacteria, colony PCR verification and sequencing confirmed that the engineered Serratia marcescens strain was obtained.

4. The application of the engineered Serratia marcescens strain as described in claim 1 in increasing the yield of serotonin.

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