Tylosin industrial high-yield strain as well as construction method and application thereof

Abstract

The invention belongs to the field of microbial genetic engineering. The invention particularly relates to a tylosin industrial high-yield strain as well as a construction method and application thereof. The sequence of the ALDH interfering gene is as shown in SEQ ID NO: 3. The industrial high-yield strain of tylosin is obtained by regulating and controlling the ALDH gene of an original strain. The strain is applied to fermentation production of tylosin and related derivatives thereof. The CRISPR i technology is utilized to inhibit in streptomyces fradiae to regulate and control ALDH gene expression, the gene is knocked out by using the homologous recombination technology, and then an engineering strain for high-yield tylosin is obtained. The engineering strain can improve the fermentation yield of tylosin by 6.8% and 9.9%.

Description

Technical Field

[0001] The present invention belongs to the field of microbial genetic engineering and specifically relates to a tylosin industrial high-yield strain and its construction method and application. Background Art

[0002] Tylosin has broad-spectrum antimicrobial properties, exhibiting significant antibacterial activity against a wide range of Gram-positive and some Gram-negative bacteria. It can also promote animal growth. Due to its low toxicity, high efficacy, zero residue, and lack of cross-resistance with humans, tylosin is widely used in the livestock industry. Market demand for tylosin is enormous, but domestic companies face numerous challenges in production, particularly high production costs. This not only limits the product's market competitiveness but also hinders the development of the entire industry. Under such high cost pressures, improving production efficiency and reducing costs have become urgent challenges for domestic tylosin manufacturers.

[0003] Currently, industrially produced tylosin is generally synthesized through the secondary metabolic pathway of Streptomyces flexneri. The synthesis of this secondary metabolite is often restricted by a complex regulatory network. Previously published studies on S. flexneri have mostly focused on genes in the tylosin biosynthetic gene cluster. However, current genomic analyses indicate that genes outside the tylosin biosynthetic gene cluster also regulate tylosin biosynthesis. However, existing research has focused less on regulatory genes outside the tylosin biosynthetic gene cluster, and even fewer targets have been identified for improving tylosin production. Summary of the Invention

[0004] The purpose of the present invention is to provide a tylosin industrial high-yield strain and a construction method and application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A sequence that interferes with the ALDH gene, wherein the sequence that interferes with the ALDH gene is represented by the base sequence of SEQ ID NO: 3.

[0006] An application of the sequence for interfering with the ALDH gene, and an application of the sequence for interfering with and inhibiting the transcription of the ALDH gene.

[0007] A tylosin industrial high-yield strain can be obtained by regulating the ALDH gene of a starting strain.

[0008] The starting strain is Streptomyces freundii (commercially available known strain).

[0009] The strain utilizes the interference sequence to interfere with or knock down the ALDH gene of Streptomyces flexneri.

[0010] The ALDH gene (acetaldehyde dehydrogenase gene) base sequence is shown in SEQ ID NO: 1.

[0011] A method for constructing an industrial high-yield tylosin strain utilizes CRISPR i (CRISPR interference) technology and homologous recombination knockout technology to regulate the expression of the ALDH gene in the starting strain to construct an engineered strain.

[0012] The regulation is to interfere with the interference sequence or to knock out the ALDH gene of the starting strain using homologous recombination technology.

[0013] Specifically: Scheme 1: Using CRISPR i technology to reversibly inhibit the expression of the ALDH gene, using the interference sequence to interfere with the ALDH gene sequence, the ALDH gene sequence is shown in SEQ ID NO.1: The method includes constructing an ALDH gene interference plasmid and transferring the plasmid into a Streptomyces flexneri strain.

[0014] The interfering plasmid can be pSET-dCas9, pIJ773, or pKC1139-derived interfering plasmid. In the present invention, pSET-dCas9 (purchased from Addgene) is preferred.

[0015] SEQ ID NO.1: The amino acid sequence of the ALDH gene is shown in SEQ ID NO.2: SEQ ID NO.2: MKAHDGMYIDGDWRPAAGQDTIAVVNPADESVIGTVPAGTAEDVDAAVRAARAALPGWAATPPAERAAHLKALHAALDARRDEIAETVTAELGSPLKFSQAVHAAVPVAVAASYAELA ATHPFEEKVGNSTVLSEPVGVVGAITPWNYPLHQIVAKAAPAFAAGCTVVLKPAEDTPLVARLFAEIVHEAGLPAGVFNLVTGLGPVAGQALAEHPGVDMVSFTGSTGVGKRIAATAA AAVKPAALELGGKSANVILPDADLARAVNVGVANVFANSGQTCSAWTRMLVDAARYEEAVALAAAAAAKYVPGDPLDPASRLGPVVSARQRDRVTGYIERGVEEGARLVAGGPGAPEG RETGYYIRPTVFADVTPDMTIAQEEIFGPVLSILKYEDEEEAVAIANGTVYGLAGAVWSADTERAAAFARRLETGQVDINGGRFNPLAPFGGYKQSGVGRELGSHGLAEYLQTKSLQF Solution 2: Knockout the ALDH gene using homologous recombination technology, the method comprising constructing an ALDH gene knockout plasmid, and transforming the recombinant plasmid into Streptomyces flexneri GS1, and screening for double-crossover recombinants.

[0016] The knockout plasmid may be pKC1139, pJTU1278, pOJ260, or pIJ12742, and pIJ12742 (purchased from Addgene) is preferred in the present invention.

[0017] The plasmid can be integrated into the genome of Streptomyces flexneri through conjugation transfer technology, electroporation transformation, chemical transformation and other technologies. An application of the tylosin industrial high-yield strain, and the application of the strain in the fermentation production of tylosin and related derivatives.

[0018] A method for producing tylosin comprises fermenting the strain to obtain tylosin.

[0019] The fermentation conditions of the genetically engineered Streptomyces are as follows: the obtained strain is inoculated into TSB seed liquid culture medium, cultured on a shaking table at 28°C for 72 hours, and then the culture liquid is inoculated into TYL fermentation medium at a rate of 4wt%, and cultured on a shaking table at 28°C for 7 days, to obtain high-yield and high-purity tylosin A.

[0020] The advantages of the present invention are: The present invention utilizes CRISPR i technology to inhibit and regulate ALDH gene expression in Streptomyces flexneri and uses homologous recombination to knock out the gene, thereby obtaining an engineered strain that produces high tylosin yields. This engineered strain can increase tylosin fermentation yields by 6.8% and 9.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC detection profiles and yield comparison histograms of the fermentation broths of the interfering strain GS1-pSET-dCas9-ALDH and the starting strain GS1 provided in an embodiment of the present invention; Figure A is an HPLC detection profile of the fermentation broths of the engineered strain GS1-pSET-dCas9-ALDH and the starting strain GS1, and Figure B is a comparison of the tylosin content in the fermentation broths of the engineered strain GS1-pSET-dCas9-ALDH and the starting strain GS1.

[0022] Figure 2 This is the electrophoresis diagram of the PCR verification of the single-crossover strain provided in the embodiment of the present invention, wherein 1-6 correspond to the six single colonies picked, the genome control is the genome of Streptomyces flexneri GS1, and the plasmid control is the pIJ12742-ALDH plasmid.

[0023] Figure 3 This is an electrophoresis diagram of PCR verification of the double-crossover strain provided in an embodiment of the present invention, wherein the genome control is the Streptomyces flexneri GS1 genome, and the plasmid control is the pIJ12742-ALDH plasmid.

[0024] Figure 4 HPLC detection profiles and yield comparison histograms of fermentation broths of the knockout strain GS1-pIJ12742-ALDH and the starting strain GS1 provided in an embodiment of the present invention; Figure A is an HPLC detection profile of the fermentation broths of the engineered strain GS1-pIJ12742-ALDH and the starting strain GS1, and Figure B is a comparison of the tylosin content in the fermentation broths of the engineered strain GS1-pIJ12742-ALDH and the starting strain GS1. DETAILED DESCRIPTION

[0025] The present invention is further described below by specific examples, which will help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. This study first discovered that the ALDH gene in Streptomyces flexneri significantly inhibits the synthesis of tylosin secondary metabolites. The ALDH gene is a negative regulator of tylosin production in S. flexneri. During the fermentation process of S. flexneri, ALDH may catalyze the oxidation of aldehyde-containing compounds to form corresponding carboxylic acids. These carboxylic acids may affect the pH of the intracellular microenvironment, thereby inhibiting tylosin production. This study provides a novel gene target, the ALDH gene, that can increase tylosin production in S. flexneri. Targeting this gene can derepress metabolic pathways, providing a new strategy for developing high-yield strains.

[0026] Furthermore, a plasmid that inhibits or knocks out the expression of the ALDH gene is introduced into a Streptomyces flexneri strain to obtain a high-yield engineered strain.

[0027] The strains involved in the following examples were obtained from purchased industrial Streptomyces flexneri (for its gene information, see Streptomyces sp. strain SL49 16S ribosomal RNA gene, partial sequence, Sequence ID: MN818640.1), as shown in Table 1.

[0028] Table 1

[0029] Example 1: ALDH gene interference high-yield strain constructed using CRISPRi technology 1. Construction of ALDH gene interference plasmid pSET-dCas9-ALDH First, deep sequencing of the transcriptome of Streptomyces flexneri GS1 at different growth stages was performed to determine the transcriptional profile of all genes in the starting strain. Subsequently, this information was collated and compared with tylosin fermentation levels to identify hundreds of target genes potentially impacting tylosin fermentation production. To validate the effects of these unknown genes, CRISPRi technology was used to disrupt their expression. Following the sgRNA design method described in Hsu, P., Scott, D., Weinstein, J. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31, 827–832 (2013), hundreds of target sites were designed and screened for fermentation. Ultimately, CRISPRi interference of the ALDH gene was confirmed to increase tylosin fermentation yield, identifying it as a potential target and designating it as ALDH-sgRNA. Its DNA sequence is shown in SEQ ID NO. 3.

[0030] Table 2 DNA sequences for transcribed sgRNA

[0031] The chemically synthesized DNA fragment (SEQ ID NO. 3) was cloned strictly in the 5'-3' direction between the Spe I and Eco RI sites of the pSET-dCas9 vector to obtain an sgRNA expression vector named pSET-dCas9-ALDH (the vector was sent to Suzhou GeneWeizhi Biotechnology Co., Ltd. for synthesis).

[0032] 2. Preparation of Donor Bacteria The plasmid pSET-dCas9-ALDH obtained above was introduced into Escherichia coli ET12567 by chemical transformation to screen transformants with apramycin resistance as plasmid donor bacteria.

[0033] 3. Preparation of Recipient Bacteria Fresh Streptomyces flexneri spores (GS1) were washed with physiological saline to prepare a spore suspension as the recipient bacteria.

[0034] 4. Conjugation transfer The plasmid donor bacteria obtained above were mixed with the recipient bacterial solution at an appropriate ratio (typically a 1:1 ratio by weight). The mixture was evenly spread on MS solid medium and incubated at 28°C for 16 hours. During this process, the donor bacteria and the recipient bacteria transferred the vector from the donor to the recipient through conjugation. After the incubation period, the plates were overlaid with a mixture of 25 μg / mL nalidixic acid and 50 μg / mL apramycin. Single colonies that successfully underwent conjugation were screened for resistance, resulting in the single strain (GS1 / pSET-dCas9-ALDH).

[0035] MS solid medium (g / L): 20g soybean meal, 20g mannitol, and 20g agar per liter of water. Adjust the pH to 7.0-7.4 and sterilize by autoclaving at 115°C for 15 minutes.

[0036] 5. Fermentation Directly dig out 1cm of GS1 / pSET-dCas9-ALDH interference strain agar block 2 Inoculate into Tylosin seed culture medium, with Streptomyces freundii GS1 as a control, and culture for 48 h. Pipette 5 mL of seed liquid and transfer to a 500 mL Erlenmeyer flask containing 50 mL of fermentation medium. Set up 3 parallel samples for each strain, culture at 28°C, 220 rpm, and culture for 7 days.

[0037] Tylos Seed Medium (g / L): 8g soybean cake powder, 2.5g yeast extract, 4g corn steep liquor, 4g calcium carbonate, and 4g soybean oil per liter of water. Adjust the pH to 6.7-6.9, incubate at 28±1°C, rotate at 220 rpm, and incubate for 48 hours.

[0038] Fermentation medium (g / L): 15g corn starch, 10g corn gluten meal, 13g cottonseed protein meal, 0.62g betaine hydrochloride, 0.5g diammonium phosphate, 0.9g potassium chloride, 1.0g sodium chloride, 20g calcium carbonate, and 45g soybean oil per liter of water. Adjust the pH to 7.1-7.3, incubate at 28±1°C, rotate at 220 rpm, and incubate for 6 days.

[0039] 6.HPLC detection Weigh 5 g of each fermentation broth into a centrifuge tube, add 5 mL of ethanol, mix thoroughly, sonicate for 30 minutes, then add 40 mL of ddH₂O, mix thoroughly, centrifuge at 8000 rpm for 10 minutes, filter the supernatant through a 0.22 μm filter, and analyze the reaction using HPLC. Each reaction was repeated three times, with a control group included. Tylosin fermentation levels were analyzed using an Agilent HPLC instrument and an Agilent-C18 column.

[0040] HPLC analysis conditions: Agilent C18 column (4.6 × 150 mm, 5 μm) using an Agilent HPLC platform; mobile phase A: 2 M sodium perchlorate solution; mobile phase B: acetonitrile, 3:2 ratio, pH 2.5. Flow rate: 1 mL / min, column temperature: 30°C, injection volume: 20 μL, detection wavelength: 280 nm.

[0041] 7. Results: According to the above HPLC test results (such as Figure 1 Figure 1 shows a comparison of tylosin content in the ALDH gene-interference strain GS1 / pSET-dCas9-ALDH and the starting strain (GS1). The GS1 / pSET-dCas9-ALDH fermentation titer reached 13,713 μg / mL, a 6.8% increase compared to the 12,843 μg / mL titer of GS1. This result demonstrates that interfering with ALDH gene expression effectively improves fermentation performance and increases tylosin production in Streptomyces freundii.

[0042] Example 2 Construction of ALDH gene knockout strain Based on the positive results of CRISPRi, a homologous recombination knockout vector was constructed to permanently knock out the gene. After confirming the positive results that CRISPRi technology can inhibit ALDH gene expression to improve the fermentation performance of Streptomyces freundii, the following steps were taken: 1. Construction of ALDH gene knockout strain pIJ12742-ALDH Bioinformatics software was used to analyze the upstream and downstream sequences of the ALDH gene and design homology arms. The length of the upstream and downstream homology arms was selected to be 3 kb each to ensure efficient and accurate homologous recombination in cells.

[0043] Using the genome of Streptomyces flexneri GS1 as a template, PCR amplification was performed with primers pIJ12742-ALDH-FF (SEQ ID NO. 4) and ALDH-FR (SEQ ID NO. 5) to obtain the upstream homology arm fragment of the ALDH gene, which was approximately 3 kb in size.

[0044] Table 3 Primer sequences of upstream homology arms of ALDH gene

[0045] The upstream homology arm fragment was ligated with the linearized plasmid fragment of plasmid pIJ12742 digested with EcoR I and Xba I by Gibson assembly to generate the recombinant plasmid pIJ12742-ALDH-F. The recombinant plasmid pIJ12742-ALDH-F was verified to be correct by sequencing.

[0046] A similar construction strategy was used to amplify the genome of Streptomyces flexneri GS1 using primers ALDH-RF (SEQ ID NO. 6) and pIJ12742-ALDH-RR (SEQ ID NO. 7) to obtain the downstream homology arm fragment of the ALDH gene, which was approximately 3 kb in size.

[0047] Table 4 Primer sequences for downstream homology arms of ALDH genes

[0048] The downstream homology arm fragment was ligated with the linearized plasmid fragment of plasmid pIJ12742-ALDH-F digested with Xba I and Hind III using the Gibson assembly method to generate the ALDH gene-deficient recombinant plasmid pIJ12742-ALDH. The recombinant plasmid pIJ12742-ALDH was verified to be correct by sequencing.

[0049] 2. Preparation of Donor Bacteria The correctly verified knockout plasmid pIJ12742-ALDH was introduced into Escherichia coli ET12567 by chemical transformation to screen transformants with apramycin resistance as plasmid donor bacteria.

[0050] 3. Preparation of Recipient Bacteria Fresh Streptomyces flexneri spores (GS1) were washed with physiological saline to prepare a spore suspension as the recipient bacteria.

[0051] 4. Conjugation transfer Mix the plasmid donor bacteria obtained above with the recipient bacteria obtained above at an appropriate ratio (typically a 1:1 ratio, weight percent). Spread the mixture evenly on conjugation transfer medium and incubate at 28°C for 16 hours. During this process, the donor and recipient bacteria transfer the vector from the donor to the recipient through conjugation. After the incubation period, overlay the plates with a mixture of 25 μg / mL nalidixic acid and 50 μg / mL apramycin. Screen for single colonies that have successfully undergone conjugation transfer based on resistance.

[0052] 5. Single-swap strain verification Six single colonies (marked 1-6) were selected for further colony PCR verification to confirm whether the vector was successfully integrated into the recipient bacterial genome and a single crossover occurred. The single crossover strain to be screened was amplified by PCR using primers ALDH-JCF (SEQ ID NO.8) / ALDH-JCR (SEQ ID NO.9). The electrophoresis results were as follows: Figure 2 As shown (i.e., single colonies 1-6 correspond to lanes 1-6), the strain with double bands of 1.9 kb and 0.5 kb amplified products in lanes 2, 3, 4, and 5 is the pIJ12742-ALDH single-exchange strain (pIJ12742-ALDH plasmid was PCR amplified to obtain a DNA product of 0.5 kb, and the Streptomyces flexneri GS1 genome was PCR amplified to obtain a DNA product of 1.9 kb). This result is consistent with expectations, indicating that a single crossover occurred in the plasmid.

[0053] Table 5 Primer sequences for verification of single-crossover strains

[0054] 6. Double-exchange strain screening Prepare two different screening media, one is a MS solid medium plate containing 50μg / mL apramycin, and the other is a MS solid medium plate without antibiotics. Figure 2 The single colony No. 3 corresponding to lane 3 in the middle was verified to be the strain with single exchange by PCR) and diluted and divided. After the culture was completed, the single colony was picked with a sterilized toothpick and spotted onto a resistance plate and a non-resistance plate respectively, and cultured at 28°C for 3 days. The growth of the colonies on the plates was observed.

[0055] 7. Double-swap strain verification Colonies that grow on both plates indicate they contain the vector-integrated genome. Colonies that grow on the antibiotic-free plate but not on the resistance plate indicate strains that have lost the resistance gene. Therefore, a double crossover has occurred, eliminating the resistance gene from the plasmid and retaining the inserted fragment. Through experimental manipulation, a strain that grows on the antibiotic-free plate but not on the resistance plate was identified. This strain was then further validated by molecular biology (for methods described in Cullum, John; Wolf, Kerstin; Doležal, Anamarija; Hranueli, Daslav The use of homologous recombination for cloning secondary metabolite clusters in Streptomyces / / Final Programme and Book of Abstracts. Zagreb: Hrvatsko genetičko društvo, 2005. str. 40 (PL-22)-x). The double-crossover strain to be screened was amplified by PCR using primers ALDH-JCF / ALDH-JCR. The size of the amplified product was used to preliminarily determine whether homologous recombination occurred. The electrophoresis results were as follows: Figure 3 The double-crossover strain amplified product was 0.5 kb in size, confirming that the strain was a pIJ12742-ALDH double-crossover strain. Correspondingly, PCR amplification of the pIJ12742-ALDH single-crossover strain yielded DNA products of 1.9 kb and 0.5 kb. PCR amplification of the pIJ12742-ALDH plasmid yielded a 0.5 kb DNA product. PCR amplification of the Streptomyces flexneri GS1 genome (genomic control) yielded a 1.9 kb DNA product, consistent with expectations.

[0056] 8. Fermentation Directly dig out 1cm of agar block of GS1 / pIJ12742-ALDH double exchange strain 2 Inoculate into Tylosin seed culture medium, using Streptomyces freundii GS1 as a control, and culture for 46-48 hours. Pipette 5 mL of seed solution and transfer to a 500 mL Erlenmeyer flask containing 50 mL of fermentation medium. Set up 3 parallel samples for each strain, culture at 28°C, 220 rpm, and culture for 7 days.

[0057] Tylos Seed Medium (g / L): 8g soybean cake powder, 2.5g yeast extract, 4g corn steep liquor, 4g calcium carbonate, and 4g soybean oil per liter of water. Adjust the pH to 6.7-6.9, incubate at 28±1°C, rotate at 220 rpm, and incubate for 48 hours.

[0058] Fermentation medium (g / L) contains 15g corn starch, 10g corn gluten meal, 13g cottonseed protein meal, 0.62g betaine hydrochloride, 0.5g diammonium phosphate, 0.9g potassium chloride, 1.0g sodium chloride, 20g calcium carbonate, and 45g soybean oil per liter of water. Adjust the pH to 7.1-7.3, incubate at 28±1°C, rotate at 220 rpm, and incubate for 6 days.

[0059] 9.HPLC detection Weigh 5 g of fermentation broth from the double-exchange strain into a centrifuge tube, add 5 mL of ethanol, mix thoroughly, sonicate for 30 minutes, then dilute with 40 mL of purified water, shake end over end, centrifuge at 8000 rpm for 10 minutes, filter the supernatant through a 0.22 μm filter, and analyze the reaction using HPLC. Each reaction was repeated three times, with a control group included. Tylosin fermentation levels were analyzed using an Agilent HPLC instrument and an Agilent-C18 column.

[0060] HPLC analysis conditions: Agilent C18 column (4.6 × 150 mm, 5 μm) using an Agilent HPLC platform; mobile phase A: 2 M sodium perchlorate solution; mobile phase B: acetonitrile, 3:2 ratio, pH 2.5. Flow rate: 1 mL / min, column temperature: 30°C, injection volume: 20 μL, detection wavelength: 280 nm.

[0061] 10. Results: Based on the HPLC test results, the tylosin content in the ALDH gene knockout strain GS1 / pIJ12742-ALDH was compared with the starting strain GS1. The tylosin titer of the GS1 / pIJ12742-ALDH strain reached 14,117 μg / mL at the end of fermentation, a 9.9% increase compared to the 12,843 μg / mL titer of GS1. These results indicate that knocking out the ALDH gene effectively improves the fermentation performance of Streptomyces freundii and increases the yield of the target product. Figure 4 ).

[0062] In summary, by regulating the expression level of the ALDH gene in Streptomyces flexneri, the present invention obtained the ALDH gene interference strain GS1 / pSET-dCas9-ALDH and the knockout strain GS1 / pIJ12742-ALDH, which increased the tylosin production by 6.8% and 9.9%, respectively, compared with the starting strain.

Claims

1. A sequence that interferes with the ALDH gene, characterized in that: The sequence of the ALDH gene interference is shown in SEQ ID NO:

3.

2. A use of the sequence for interfering with the ALDH gene according to claim 1, characterized in that: The application of the sequence in interfering with and inhibiting the transcription of the ALDH gene.

3. A tylosin industrial high-yield strain, characterized in that: This strain is obtained by regulating the ALDH gene of the starting strain.

4. The tylosin industrial high-yield strain according to claim 3, characterized in that: The starting strain is Streptomyces freundii.

5. The tylosin industrial high-yield strain according to claim 3 or 4, characterized in that: The strain utilizes the ALDH gene interfering sequence according to claim 1 to interfere with the ALDH gene of Streptomyces flexneri.

6. The tylosin industrial high-yield strain according to claim 3, characterized in that: The ALDH gene sequence is shown in SEQ ID NO:

1.

7. A method for constructing the tylosin industrial high-yield strain according to claim 3, characterized in that: CRISPRi technology and homologous recombination knockout technology were used to regulate the expression of ALDH gene in the starting strain and construct the engineered strain.

8. The method for constructing the tylosin industrial high-yield strain according to claim 7, characterized in that: The regulation is to interfere with the ALDH gene using the sequence of claim 1 or to knock out the ALDH gene of the starting strain using homologous recombination knockout technology.

9. An application of the tylosin industrial high-yield strain according to claim 3, characterized in that: The strain is used in fermentation production of tylosin and related derivatives.

10. A method for producing tylosin, characterized in that: Tylosin is obtained by fermenting the tylosin industrial high-yield strain according to claim 3.

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