Method for increasing yield of indole-3-acetic acid through streptomyces scabies SCAB31831 gene and application

Abstract

The invention provides a method for increasing the yield of indole-3-acetic acid through a streptomyces scabies SCAB31831 gene, which comprises the following steps: deleting the SCAB31831 gene in streptomyces scabies through a genetic engineering way to obtain an indole-3-acetic acid high-yield engineering strain, and fermenting the strain to produce the indole-3-acetic acid. Wherein the sequence of the SCAB31831 gene is as shown in SEQ ID NO. 1. The invention also provides an application of the method in preparation of an indole-3-acetic acid high-yield microbial preparation, and provides an indole-3-acetic acid high-yield freeze-dried bacterial powder. According to the invention, the SCAB31831 gene negatively related to the biosynthesis of the indole-3-acetic acid is identified, and the gene is deleted in streptomyces scabies, so that the indole-3-acetic acid high-yield strain and freeze-dried bacterial powder based on the strain are obtained, and a technical support is provided for improving the fermentation yield of the indole-3-acetic acid in industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method and application of increasing the yield of indole-3-acetic acid by using the gene of Streptomyces scabii SCAB_31831. Background Art

[0002] Streptomyces is a Gram-positive actinomycete widely distributed in soil. Within ecosystems, it decomposes organic matter and maintains soil health. Furthermore, due to the rich variety and structural diversity of its secondary metabolites, Streptomyces has become an important resource for natural drug development. Streptomyces' secondary metabolites are controlled by a complex, multi-layered regulatory network, involving multi-dimensional regulatory mechanisms such as the coordinated expression of genes encoding key metabolic pathway enzymes, the cascade activation of regulatory factors, and the dynamic allocation of precursor metabolism. Metabolic pathway-related genes dynamically regulate the biosynthesis of secondary metabolites by coordinating the spatiotemporal expression of secondary metabolic gene clusters with the allocation of precursor metabolism. Therefore, targeted modification of metabolic pathway genes can be performed to enhance the allocation of precursors for target metabolites, thereby achieving high yields of target secondary metabolites. For example, targeted genetic modification through genetic engineering has resulted in a Streptomyces strain that produces high indole-3-acetic acid for use in the production of indole-3-acetic acid.

[0003] Indole-3-acetic acid (IAA) is an important plant hormone used as a growth regulator in agriculture, promoting rooting, improving stress resistance, and increasing yield. However, IAA's chemical instability and low yield lead to high costs and a short shelf life for its commercial formulations. Therefore, developing methods for high-yield and stable storage of IAA is of great significance for agricultural production.

[0004] In Streptomyces scabies, indole-3-acetic acid is primarily synthesized via the tryptophan-dependent indole-3-acetamide pathway. In this pathway, tryptophan is catalyzed by tryptophan-2-monooxygenase to produce indoleacetamide, which is then decomposed into indole-3-acetic acid by indole-3-acetamide hydrolase. Furthermore, in Streptomyces scabies, tryptophan is a common precursor for the biosynthesis of indole-3-acetic acid and another secondary metabolite, thaxtomin A. The SCAB_31831 gene encodes a cytochrome P450 monooxygenase responsible for catalyzing the first step in thaxtomin A biosynthesis, converting tryptophan to L-4-nitrotryptophan. Therefore, the present inventors speculate that deletion of the SCAB_31831 gene can block the conversion of tryptophan to the thaxtomin A biosynthetic pathway, thereby increasing the availability of precursor raw materials for the indole-3-acetic acid biosynthetic pathway.

[0005] However, there is still a lack of research on the metabolic distribution mechanism of precursors for the biosynthesis of indole-3-acetic acid and thaxtomin A in Streptomyces scabies. There is also no research on the modification of the tryptophan metabolic pathway through genetic engineering to increase the production of indole-3-acetic acid in Streptomyces scabies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method and application for increasing the production of indole-3-acetic acid by using the gene of Streptomyces scabii SCAB_31831. The method obtains a method for increasing the production of indole-3-acetic acid by exploring the relationship between the gene of Streptomyces scabii SCAB_31831 and the biosynthesis of indole-3-acetic acid.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A method for increasing the yield of indole-3-acetic acid by using the SCAB_31831 gene of Streptomyces scabii. The method comprises deleting the SCAB_31831 gene in Streptomyces scabii through genetic engineering to obtain an engineered strain with high indole-3-acetic acid yield, and then using the obtained strain to ferment and produce indole-3-acetic acid. The nucleotide sequence of the SCAB_31831 gene is shown in SEQ ID NO.1.

[0009] As one preferred embodiment of the present invention, in the step of deleting the SCAB_31831 gene in Streptomyces scabii via genetic engineering, the Streptomyces scabii strain 87.22 is used. The Streptomyces scabii strain 87.22 is a publicly available strain provided by the China General Microbiological Culture Collection Center with a deposit number of CGMCC 4.1765.

[0010] As one of the preferred embodiments of the present invention, the expression product of the SLCG_3904 gene is used to negatively regulate the biosynthesis of indole-3-acetic acid.

[0011] As one of the preferred embodiments of the present invention, the SCAB_31831 gene is used to encode cytochrome P450 monooxygenase; the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO. 2, which is responsible for catalyzing the conversion of tryptophan to L-4-nitrotryptophan (the first step in thaxtomin A biosynthesis);

[0012] By deleting the SCAB_31831 gene through genetic engineering, the conversion of tryptophan to L-4-nitrotryptophan can be blocked, thereby promoting the biosynthesis of indole-3-acetic acid.

[0013] The invention relates to an application of the above method in preparing a microbial preparation with high indole-3-acetic acid production.

[0014] The invention discloses a freeze-dried bacterial powder for producing high-yield indole-3-acetic acid. The invention comprises the following steps: firstly, the SCAB_31831 gene in Streptomyces scabii is deleted through genetic engineering to obtain an engineered strain for producing high-yield indole-3-acetic acid, wherein the nucleotide sequence of the SCAB_31831 gene is shown in SEQ ID NO. 1; secondly, 10 mM tryptophan is added to the fermentation medium of the engineered strain for producing high-yield indole-3-acetic acid, and the strain is fermented and cultured; and finally, the obtained fermentation broth is freeze-dried to produce bacterial powder.

[0015] As one of the preferred embodiments of the present invention, the final fermentation medium of the indole-3-acetic acid high-yield engineered strain is: OBB liquid medium (oat bran medium) + 10 mM tryptophan.

[0016] As one of the preferred embodiments of the present invention, after the fermentation of the indole-3-acetic acid high-yield engineered strain is completed, a protective agent is added to the fermentation broth, which is then freeze-dried and ground into powder.

[0017] As one of the preferred embodiments of the present invention, the protective agent comprises: 3% glycerol, 1% ascorbic acid, 5% mannitol, and 1% sodium dihydrogen phosphate.

[0018] The advantages of the present invention over the prior art are:

[0019] In the present invention, a cytochrome P450 monooxygenase gene SCAB_31831, which is negatively correlated with indole-3-acetic acid biosynthesis, was identified. The SCAB_31831 gene was deleted in Streptomyces scabii through genetic engineering to obtain an indole-3-acetic acid high-yielding strain (i.e., a Streptomyces scabii SCAB_31831 gene-deleted mutant). The fermentation broth of the indole-3-acetic acid high-yielding strain was freeze-dried to prepare bacterial powder, providing technical support for increasing the fermentation yield of indole-3-acetic acid in industrial production and for the agricultural application of freeze-dried bacterial powder rich in indole-3-acetic acid.

[0020] Among them, when the SCAB_31831 gene was deleted in Streptomyces scabii, the indole-3-acetic acid production increased to 2.8 times that of the original strain; after the SCAB_75421 gene was complemented in the ΔSCAB_31831 mutant, the indole-3-acetic acid production was restored; this indicates that there is a negative correlation between the SCAB_31831 gene and indole-3-acetic acid biosynthesis.

[0021] Furthermore, the present invention found that adding 10 mM tryptophan to the culture medium of the ΔSCAB_31831 mutant significantly increased indole-3-acetic acid production to 42 times that of the original strain, demonstrating that the SCAB_31831 gene deletion combined with the addition of the precursor tryptophan can be used to specifically enhance indole-3-acetic acid biosynthesis in Streptomyces scabies. Finally, freeze-drying the indole-3-acetic acid-rich fermentation broth significantly improved its stability, effectively preserving its biological activity and extending its shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the structure of the pUCTSR plasmid used in the present invention.

[0023] Figure 2 Schematic diagram of the construction process of the SCAB_31831 gene deletion mutant strain ΔSCAB_31831 of the present invention (using the suicide plasmid pUCTSR and homologous recombination technology to delete 982 bp within the SCAB_31831 gene on the chromosome of Streptomyces scabii);

[0024] Figure 3 This is a PCR verification image of the mutant strain ΔSCAB_31831 of the present invention (in the figure, "M": 5000 bp DNA Marker; "1": positive control, 1599 bp; "2": negative control, 1221 bp; "3": PCR amplification band of the positive clone, the same as "1");

[0025] Figure 4 This is a PCR verification image of the complemented strain ΔSCAB_31831 / pIB-31831 of the present invention (in the image, "M": 5000 bp DNA marker; "1": internal fragment of the apr resistance gene on the pIB139 plasmid, i.e., 750 bp positive control; "2, 3": PCR amplification bands of the screened positive clones, the same as "1");

[0026] Figure 5 HPLC analysis of indole-3-acetic acid production in fermentation broths of the starting strain 87.22, the mutant strain ΔSCAB_31831, the complemented strain ΔSCAB_31831 / pIB-31831, and the empty vector control strain ΔSCAB_31831 / pIB139 of the present invention (in the figure, "***": P < 0.001);

[0027] Figure 6Graphs showing transcriptional analysis of genes related to indole-3-acetic acid biosynthesis in the ΔSCAB_31831 mutant of the present invention (Figure A shows a transcriptional analysis of the indole-3-acetic acid biosynthesis gene iaaH; Figure B shows a transcriptional analysis of the indole-3-acetic acid biosynthesis gene iaaM);

[0028] Figure 7 HPLC analysis of indole-3-acetic acid production in the fermentation broth after adding different concentrations of tryptophan to the starting strain 87.22 and the mutant strain ΔSCAB_31831 of the present invention;

[0029] Figure 8 This is a physical picture of the freeze-dried bacterial powder rich in indole-3-acetic acid of the present invention (in the figure, Figures A and B are physical pictures from different perspectives respectively). DETAILED DESCRIPTION

[0030] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0031] The strains and plasmids used in the following examples are shown in Table 1, and the primer sequences used are shown in Table 2.

[0032] Escherichia coli used in the following examples was cultured in liquid LB medium or on LB plates supplemented with 2.0% agar at 37°C; Streptomyces scabies 87.22 was cultured in TSBY medium or on SFM plates supplemented with 2% agar at 28°C. When Streptomyces scabies was fermented to produce indole-3-acetic acid, OBB liquid fermentation medium (oat bran medium) was used and cultured at 28°C. General techniques for E. coli and Streptomyces scabies were performed according to standard procedures. Primer synthesis and DNA sequencing were performed by Universal Biosystems (Anhui) Co., Ltd.

[0033] Liquid LB medium formula: 10 g tryptone, 5 g yeast extract, 10 g NaCl, distilled water to 1000 mL, and adjust the pH to 7.0.

[0034] LB solid plate medium (added with 2.0% agar) formula: liquid LB medium + 2.0% agar.

[0035] TSBY medium formula: 15 g tryptone, 5 g soy peptone, 5 g NaCl, dilute to 1000 mL with distilled water, and adjust the pH to 7.2.

[0036] SFM solid plate medium (containing 2% agar) formula: 20 g of soybean cake powder, 800 mL of tap water, filter and remove the supernatant after sterilization, add 20 g of mannitol, dilute to 1000 mL, adjust the pH to 7.3, package into 4 x 250 mL, and then add agar to a final concentration of 2%.

[0037] Formula of OBB liquid fermentation medium (oat bran medium): 30g oat bran, 1000mL tap water, bring to a boil and then simmer on low heat for 10min, stirring while boiling, filter out impurities with gauze, dilute the filtrate to 1000mL, and divide into 50mL culture medium.

[0038] Table 1 The present invention relates to bacterial strains and plasmids

[0039]

[0040]

[0041] Table 2 Primers of the present invention

[0042]

[0043]

[0044] Example 1

[0045] SCAB_31831 gene related information:

[0046] The nucleotide sequence of the SCAB_31831 gene is shown in SEQ ID NO. 1, which encodes cytochrome P450 monooxygenase; the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO. 2, which is responsible for catalyzing tryptophan to produce L-4-nitrotryptophan.

[0047] Example 2

[0048] Construction of gene deletion mutant of Streptomyces scabii SCAB_31831:

[0049] To construct a gene deletion mutant of Streptomyces scabii SCAB_31831, see Figure 2 The internal sequence of the SCAB_31831 gene was replaced using the thiostrepton resistance gene tsr carried by the suicide plasmid pUCTSR.

[0050] Using the Streptomyces scabii genome as a template, PCR amplified 1500-bp upstream and downstream homology arms of the SCAB_31831 gene using primers 31831-UF / R and 31831-DF / R, respectively. The primer sequences are shown in Table 2. The underlined "AAGCTT," "TCTAGA," "GGATCC," and "GAATTC" sequences represent the restriction endonuclease sites for HindIII, XbaI, KpnI, and EcoRI, respectively.

[0051] The upstream and downstream DNA fragments were cloned into the pUCTSR plasmid using the above restriction endonucleases to obtain the recombinant plasmid pUCTSR-Δ31831. The recombinant plasmid pUCTSR-Δ31831 was transformed into Escherichia coli ET12567 (pUZ8002), and pUCTSR-Δ31831 was introduced into Streptomyces scabii 87.22 by cross-genus conjugation transfer technology. The conjugates with resistance were selected using thiostrepton. The selected strains were subjected to PCR analysis using primers 31831-CF / R. The correct positive strain was named ΔSCAB_31831 ( Figure 3 ). Specific primer sequences are shown in Table 2.

[0052] Example 3

[0053] Construction of SCAB_31831 gene complementation strain:

[0054] To complement the SCAB_31831 gene in the ΔSCAB_31831 mutant, the entire DNA fragment of the SCAB_31831 gene was amplified by PCR using the S. scabii genome as a template and primers 31831-CF / R. The primer sequences are shown in Table 2. The underlined "CATATG" and "TCTAGA" sequences represent the restriction endonuclease sites NdeI and XbaI, respectively.

[0055] The SCAB_31831 gene fragment was ligated to plasmid pIB139 using the aforementioned restriction endonucleases to construct the recombinant plasmid pIB-31831. The recombinant plasmid pIB-31831 was transformed into ET12567 (pUZ8002), and pIB-31831 was introduced into the deletion mutant ΔSCAB_31831 by conjugation. Apramycin-resistant conjugates were selected, and PCR verification (using primers Apr-F / R) yielded the complemented strain ΔSCAB_31831 / pIB-31831 ( Figure 4 Using the same method, the empty vector pIB139 was introduced into the mutant strain ΔSCAB_31831 to obtain the empty vector control strain ΔSCAB_31831 / pIB139 ( Figure 4 ).

[0056] Example 4

[0057] Detection of indole-3-acetic acid production in a series of Streptomyces scabies strains:

[0058] Spores of Streptomyces scabii 87.22 and ΔSCAB_31831 strains growing on the same plate were inoculated into TSBY seed bottles and cultured at 220 rpm and 28°C for 2 days. They were then transferred to OBB liquid medium and cultured at the same speed and temperature for another 7 days. After fermentation, the indole-3-acetic acid in the fermentation broth was extracted and analyzed by HPLC. The yield of indole-3-acetic acid was calculated using an indole-3-acetic acid standard curve ( Figure 5 ).

[0059] Example 5

[0060] Transcriptional analysis of related genes in ΔSCAB_31831:

[0061] RNA was extracted from Streptomyces scabii 87.22 and mutant strain ΔSCAB_31831 during fermentation using an RNA extraction kit. RNA was converted into cDNA and the transcription levels of genes related to indole-3-acetic acid biosynthesis were analyzed using real-time quantitative PCR (RT-qPCR). Figure 6 ).

[0062] Example 6

[0063] Analysis of indole-3-acetic acid production in the ΔSCAB_31831 mutant after adding different concentrations of tryptophan:

[0064] Spores of Streptomyces scabii 87.22 and ΔSCAB_31831 growing on the same plate were inoculated into TSBY medium and shake-cultured for 2 days to serve as seed culture. The seed culture was then transferred to OBB liquid medium containing different concentrations of tryptophan and shake-cultured at 220 rpm and 28°C for 7 days. After fermentation, the indole-3-acetic acid yield in each culture medium was analyzed by HPLC ( Figure 7 ).

[0065] Example 7

[0066] Freeze-dried fermentation broth of strains rich in indole-3-acetic acid:

[0067] A freeze-drying protective agent (3% glycerol, 1% ascorbic acid, 5% mannitol, and 1% sodium dihydrogen phosphate) was added to the fermentation broth with the highest indole-3-acetic acid concentration and mixed thoroughly. Subsequently, the treated fermentation broth was pre-frozen at -80°C for 24 hours. After pre-freezing, the fermentation broth was freeze-dried using a vacuum freeze dryer at -90°C and 0.1 mbar for 72 hours and ground into powder ( Figure 8 ).

[0068] Example 8

[0069] Analysis of the results of the above embodiments:

[0070] 1. The indole-3-acetic acid production was significantly increased after the SCAB_31831 gene was deleted.

[0071] The SCAB_31831 gene deletion mutant ΔSCAB_31831 has been verified by PCR ( Figure 3 ΔSCAB_31831 was fermented in OBB liquid medium for 7 days, and after extraction and HPLC analysis, the indole-3-acetic acid production of the mutant strain ΔSCAB_31831 was significantly increased to 2.8 times that of the original strain ( Figure 5 ), indicating a negative correlation between the SCAB_31831 gene and indole-3-acetic acid biosynthesis.

[0072] 2. SCAB_31831 gene complementation.

[0073] To confirm that the phenotype of the mutant strain ΔSCAB_31831 was entirely due to the SCAB_31831 gene mutation, the present invention designed a SCAB_31831 gene complementation experiment for verification. pIB-31831 uses the strong promoter PermE* of the erythromycin resistance gene to initiate transcriptional expression of the SCAB_31831 gene for complementation of the mutant strain ΔSCAB_31831. After fermentation culture and HPLC detection, the indole-3-acetic acid production of the complemented strain ΔSCAB_31831 / pIB-31831 was restored to a level comparable to that of the original strain 87.22 ( Figure 5 ).

[0074] 3. After the SCAB_31831 gene was deleted, the transcription of indole-3-acetic acid biosynthesis genes increased significantly.

[0075] qRT-PCR results confirmed that the expression levels of the indole-3-acetic acid biosynthesis genes iaaH and iaaM in the ΔSCAB_31831 mutant increased by 5.6 times and 8.6 times at 12 h, and by 1.9 times and 1.7 times at 24 h compared with the original strain 87.22. Figure 6); indicating that the deletion of the SCAB_31831 gene can cause a significant increase in the transcription level of genes related to indole-3-acetic acid biosynthesis, thereby increasing the production of indole-3-acetic acid.

[0076] 4. Adding the precursor tryptophan to the ΔSCAB_31831 mutant strain can significantly increase the production of indole-3-acetic acid.

[0077] To further increase the yield of indole-3-acetic acid, the present invention added different concentrations of the precursor tryptophan to the fermentation medium of the ΔSCAB_31831 mutant. HPLC analysis showed that when the tryptophan addition concentration was 10 mM, the indole-3-acetic acid yield in the ΔSCAB_31831 mutant was significantly increased to 42 times that of the original strain ( Figure 7 ); indicating that the strategy of deleting the SCAB_31831 gene combined with adding the precursor tryptophan can achieve a significant increase in the production of indole-3-acetic acid.

[0078] 5. Freeze-dry the fermentation broth of the strain rich in indole-3-acetic acid.

[0079] The present invention uses a freeze-drying protective agent to improve the stability of the fermentation broth after freeze-drying. The specific ingredients are: 3% glycerol, 1% ascorbic acid, 5% mannitol and 1% sodium dihydrogen phosphate. By fully mixing the fermentation broth with the protective agent and then freeze-drying it, the fermentation broth is converted into a powder, which can significantly enhance the storage stability while effectively maintaining its biological activity ( Figure 8 ).

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for increasing the production of indole-3-acetic acid by using the gene of Streptomyces scabii SCAB_31831, characterized in that: The SCAB_31831 gene in Streptomyces scabii was deleted through genetic engineering to obtain an engineered strain with high indole-3-acetic acid production, and the obtained strain was then used to produce indole-3-acetic acid by fermentation; wherein the nucleotide sequence of the SCAB_31831 gene is shown in SEQ ID NO.

1.

2. The method for increasing the yield of indole-3-acetic acid by using the gene of Streptomyces scabii SCAB_31831 according to claim 1, characterized in that: In the step of deleting the SCAB_31831 gene in Streptomyces scabies through genetic engineering, the Streptomyces scabies used is the Streptomyces scabies 87.22 strain.

3. The method for increasing indole-3-acetic acid production by using the gene of Streptomyces scabii SCAB_31831 according to claim 1, characterized in that: The expression product of the SLCG_3904 gene is used to negatively regulate the biosynthesis of indole-3-acetic acid.

4. The method for increasing the yield of indole-3-acetic acid by using the gene of Streptomyces scabii SCAB_31831 according to claim 3, characterized in that: The SCAB_31831 gene is used to encode cytochrome P450 monooxygenase; the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO. 2, which is responsible for catalyzing tryptophan to produce L-4-nitrotryptophan; By deleting the SCAB_31831 gene through genetic engineering, the conversion of tryptophan to L-4-nitrotryptophan can be blocked, thereby promoting the biosynthesis of indole-3-acetic acid.

5. Use of the method according to any one of claims 1 to 4 in the preparation of a microbial preparation with high indole-3-acetic acid production.

6. A freeze-dried bacterial powder with high yield of indole-3-acetic acid, characterized in that: First, the SCAB_31831 gene in Streptomyces scabii is deleted through genetic engineering to obtain an engineered strain that produces high indole-3-acetic acid. The nucleotide sequence of the SCAB_31831 gene is shown in SEQ ID NO.

1. Next, 10 mM tryptophan is added to the fermentation medium of the engineered strain that produces high indole-3-acetic acid, and the strain is fermented and cultured. Finally, the resulting fermentation broth is freeze-dried to produce bacterial powder.

7. The indole-3-acetic acid high-yielding freeze-dried bacterial powder according to claim 6, characterized in that: The final fermentation medium of the indole-3-acetic acid high-yield engineered strain is: oat bran medium + 10mM tryptophan.

8. The indole-3-acetic acid high-yielding freeze-dried bacterial powder according to claim 6, characterized in that: After the fermentation of the indole-3-acetic acid high-yield engineered strain is completed, a protective agent is added to the fermentation liquid, which is then freeze-dried and ground into powder.

9. The indole-3-acetic acid high-yielding freeze-dried bacterial powder according to claim 8, characterized in that The protective agent comprises: 3% glycerol, 1% ascorbic acid, 5% mannitol and 1% sodium dihydrogen phosphate.

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