Method for improving indole-3-acetic acid yield by streptomyces scabiei scab_31831 gene and application thereof

Abstract

The application provides a method for improving the yield of indole-3-acetic acid by Streptomyces scabiei SCAB_31831 gene, and the method comprises the following steps: making SCAB_31831 gene in Streptomyces scabiei to be lost through a genetic engineering approach, obtaining an indole-3-acetic acid high-yield engineering strain, and then using the strain to ferment and produce indole-3-acetic acid; wherein the sequence of the SCAB_31831 gene is shown as SEQ ID NO. 1. The application also provides application of the above 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. The application identifies the SCAB_31831 gene which is negatively related to the biosynthesis of indole-3-acetic acid, and through the loss of the gene in Streptomyces scabiei, an indole-3-acetic acid high-yield strain and a freeze-dried bacterial powder based on the strain are obtained, which provides technical support for improving the fermentation yield of indole-3-acetic acid in industrial production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method and application for increasing the production of indole-3-acetic acid using the SCAB_31831 gene of Streptomyces scabies. Background Technology

[0002] Streptomyces are a group of Gram-positive actinomycetes widely distributed in soil, playing a vital role in decomposing organic matter and maintaining soil health within ecosystems. Simultaneously, due to the rich variety and structural diversity of their secondary metabolites, Streptomyces have become an important resource for the development of natural drugs. The secondary metabolites of Streptomyces are controlled by a complex, multi-level regulatory network, involving multi-dimensional regulatory mechanisms such as the synergistic expression of genes encoding key enzymes in metabolic pathways, the cascade activation of regulatory factors, and the dynamic allocation of precursor metabolites. 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 metabolites. Therefore, targeted modification of genes in metabolic pathways can enhance the precursor allocation of target metabolites, thereby achieving high yields of target secondary metabolites. For example, by using genetic engineering to directionally alter genes, Streptomyces strains producing high levels of indole-3-acetic acid (IAA) can be obtained for the production of IAA.

[0003] Indole-3-acetic acid (IAA) is an important plant hormone used in agriculture as a growth regulator to promote root development, enhance stress resistance, and increase yield. However, IAA's chemical instability and low yield result in high costs and short shelf life for commercial formulations. Therefore, developing methods for high-yield and stably stored IAA is of great significance for agricultural production.

[0004] In *Streptomyces scabica*, indole-3-acetic acid is mainly synthesized via the tryptophan-dependent indole-3-acetamide pathway. In this pathway, tryptophan is catalyzed by tryptophan-2-monooxidase to generate indoleacetamide, which is then degraded into indole-3-acetic acid by indole-3-acetamide hydrolase. Simultaneously, in *Streptomyces scabica*, 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 inventors hypothesize that deletion of the SCAB_31831 gene can block the conversion of tryptophan to thaxtomin A, thereby allowing the indole-3-acetic acid biosynthesis pathway to obtain more precursor materials.

[0005] However, there is still a lack of research on the precursor metabolic allocation mechanism of indole-3-acetic acid and thaxtomin A biosynthesis in Streptomyces scabioides, and there are also no studies on modifying the tryptophan metabolic pathway through genetic engineering to increase the production of indole-3-acetic acid in Streptomyces scabioides. 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 SCAB_31831 gene of Streptomyces scabioides. The method for increasing the production of indole-3-acetic acid is obtained by exploring the relationship between the SCAB_31831 gene of Streptomyces scabioides and the biosynthesis of indole-3-acetic acid.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0008] A method for increasing the yield of indole-3-acetic acid (IDA) by using the SCAB_31831 gene of *Streptomyces scabica* involves deleting the SCAB_31831 gene from *Streptomyces scabica* through genetic engineering to obtain a high-yield engineered strain of IDA, which is then used to ferment and produce IDA. The nucleotide sequence of the SCAB_31831 gene is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, in the step of deleting the SCAB_31831 gene in *Streptomyces scabica* via genetic engineering, the *Streptomyces scabica* strain used is *Streptomyces scabica* strain 87.22. *Streptomyces scabica* strain 87.22 is a currently disclosed and publicly available strain, provided by the China General Microbiological Culture Collection Center, with accession number 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, and it is responsible for catalyzing the production of L-4-nitrotryptophan from tryptophan (the first step of thaxtomin A biosynthesis);

[0012] Deleting the SCAB_31831 gene through genetic engineering can block the conversion of tryptophan to L-4-nitrotryptophan and promote the biosynthesis of indole-3-acetic acid.

[0013] Application of the above method in the preparation of high-yield microbial preparations of indole-3-acetic acid.

[0014] A high-yield indole-3-acetic acid (IAA) freeze-dried bacterial powder is prepared by first deleting the SCAB_31831 gene in *Streptomyces scabica* via genetic engineering to obtain a high-yield IAA engineered strain. 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 high-yield IAA engineered strain, and the strain is fermented. Finally, the obtained fermentation broth is freeze-dried to obtain the bacterial powder.

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

[0016] As one of the preferred embodiments of the present invention, after the fermentation of the high-yield engineered strain of indole-3-acetic acid is completed, a protective agent is added to the fermentation broth, followed by freeze-drying and grinding 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 this invention compared to the prior art are:

[0019] This invention identifies the cytochrome P450 monooxygenase gene SCAB_31831, which is negatively correlated with the biosynthesis of indole-3-acetic acid (IAA). Through genetic engineering, the SCAB_31831 gene was deleted in *Streptomyces scabica*, resulting in a high-yielding IAA strain (i.e., the *Streptomyces scabica* SCAB_31831 gene deletion mutant). The fermentation broth of this high-IAA-yielding strain was freeze-dried to prepare a bacterial powder, providing technical support for increasing IAA fermentation yield in industrial production and for the agricultural application of freeze-dried bacterial powder rich in IAA.

[0020] Specifically, when the SCAB_31831 gene was missing in *Streptomyces scabica*, the production of indole-3-acetic acid increased to 2.8 times that of the original strain; however, after the SCAB_75421 gene was reintroduced into the ΔSCAB_31831 mutant strain, the production of indole-3-acetic acid was restored. This indicates that there is a negative correlation between the SCAB_31831 gene and the biosynthesis of indole-3-acetic acid.

[0021] Furthermore, this invention also found that adding 10 mM tryptophan to the culture medium of the ΔSCAB_31831 mutant strain significantly increased the yield of indole-3-acetic acid (IAA) to 42 times that of the original strain. This indicates that the SCAB_31831 gene deletion combined with the addition of precursor tryptophan can be used to directionally increase the biosynthetic yield of IAA in *Streptomyces scabica*. Finally, the stability of the fermentation broth rich in IAA was significantly improved after freeze-drying, which effectively protected its biological activity and extended its shelf life. Attached Figure Description

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

[0023] Figure 2 This is a schematic diagram of the construction process of the SCAB_31831 gene deletion mutant strain ΔSCAB_31831 of the present invention (982bp of the SCAB_31831 gene on the chromosome of Streptomyces scabica was deleted using the suicide plasmid pUCTSR and homologous recombination technology).

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

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

[0026] Figure 5 This is an HPLC chromatogram of the indole-3-acetic acid yield in the fermentation broth of the starting strain 87.22, the mutant strain ΔSCAB_31831, the complemented strain ΔSCAB_31831 / pIB-31831 and its empty vector control strain ΔSCAB_31831 / pIB139 (in the figure, "***": P<0.001);

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

[0028] Figure 7 This is an HPLC analysis chromatogram of the indole-3-acetic acid yield in the fermentation broth of the starting strain 87.22 and the mutant strain ΔSCAB_31831 after adding different concentrations of tryptophan.

[0029] Figure 8 The images shown are of the freeze-dried bacterial powder rich in indole-3-acetic acid of this invention (images A and B are images of the actual product from different perspectives). Detailed Implementation

[0030] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

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

[0033] Liquid LB medium formulation: 10g tryptone, 5g yeast extract, 10g NaCl, distilled water to a final volume of 1000mL, pH adjusted to 7.0.

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

[0035] TSBY culture medium formula: 15g tryptone, 5g soybean peptone, 5g NaCl, distilled water to a final volume of 1000mL, adjust pH to 7.2.

[0036] SFM solid plate culture medium (containing 2% agar) formula: 20g soybean meal powder, 800mL tap water, sterilize and filter to obtain the supernatant, add 20g mannitol, make up to 1000mL, adjust pH to 7.3, dispense into 4 x 250mL, and then add agar to a final concentration of 2%.

[0037] OBB liquid fermentation medium (oat bran medium) formula: 30g oat bran, 1000mL tap water, boil and then simmer for 10 minutes, stirring constantly. Filter out impurities with gauze, bring the filtrate to 1000mL, and dispense into 50mL containers.

[0038] Table 1. This invention relates to strains and plasmids.

[0039]

[0040]

[0041] Table 2 This invention relates to primers.

[0042]

[0043]

[0044] Example 1

[0045] Information related to the SCAB_31831 gene:

[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 the conversion of tryptophan to L-4-nitrotryptophan.

[0047] Example 2

[0048] Construction of the SCAB_31831 gene deletion mutant strain of Streptomyces scabioides:

[0049] To construct the SCAB_31831 gene deletion mutant strain of *Streptomyces scabii*, see [link to relevant documentation]. Figure 2 The internal sequence of the SCAB_31831 gene was replaced by the thiostreptin resistance gene tsr carried by the suicide plasmid pUCTSR.

[0050] Using the genome of *Streptomyces scabica* as a template, PCR amplification was performed on 1500 bp homologous arm DNA fragments upstream and downstream of the SCAB_31831 gene using primers 31831-UF / R and 31831-DF / R, respectively. The specific primer sequences are shown in Table 2. The underlined sequences “AAGCTT,” “TCTAGA,” “GGATCC,” and “GAATTC” represent the restriction enzyme sites of HindIII, XbaI, KpnI, and EcoRI, respectively.

[0051] The upstream and downstream DNA fragments were cloned into the pUCTSR plasmid using the aforementioned restriction endonucleases to obtain the recombinant plasmid pUCTSR-Δ31831. The recombinant plasmid pUCTSR-Δ31831 was transformed into *Escherichia coli* ET12567 (pUZ8002), and then introduced into *Streptomyces scabica* 87.22 via cross-generic conjugation transfer. Conjugators resistant to thiostreptococcal infection were screened using thiostreptococcalin. PCR analysis of the selected strains using primers 31831-CF / R confirmed the positive results, and the strain was named ΔSCAB_31831. Figure 3 The specific primer sequences are shown in Table 2.

[0052] Example 3

[0053] Construction of the SCAB_31831 gene complementation strain:

[0054] To reintroduce the SCAB_31831 gene into the ΔSCAB_31831 mutant strain, the complete DNA fragment of the SCAB_31831 gene was amplified by PCR using the *Streptomyces scabica* genome as a template and 31831-CF / R as primers. The specific primer sequences are shown in Table 2. The underlined "CATATG" and "TCTAGA" sequences are the restriction enzyme sites for NdeI and XbaI, respectively.

[0055] The SCAB_31831 gene fragment was ligated into 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 then introduced into the deletion mutant ΔSCAB_31831 via conjugation transfer. Conjugates resistant to apramycin were screened, and after PCR verification (using primers Apr-F / R), the complemented strain ΔSCAB_31831 / pIB-31831 was obtained. 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 Streptomyces strains of Streptomyces scabies:

[0058] Spores of *Streptomyces scabica* strains 87.22 and ΔSCAB_31831 with similar growth patterns on agar plates were inoculated into TSBY seed bottles and cultured with shaking at 220 rpm and 28°C for 2 days. They were then transferred to OBB liquid medium and cultured with shaking at the same speed and temperature for another 7 days. After fermentation, indole-3-acetic acid in the fermentation broth was extracted and analyzed by HPLC. The yield 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 scabica* 87.22 and the mutant strain ΔSCAB_31831 during fermentation culture using an RNA extraction kit. After being reverse-engineered into cDNA, the transcriptional 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 yield in the ΔSCAB_31831 mutant strain after adding different concentrations of tryptophan:

[0064] Spores of *Streptomyces scabica* 87.22 and ΔSCAB_31831, which exhibited similar growth on agar plates, were inoculated into TSBY medium and cultured with shaking for 2 days as seed culture medium. This seed culture medium was then transferred to OBB liquid medium containing different concentrations of tryptophan and cultured with shaking at 220 rpm and 28°C for 7 days. After fermentation, the yield of indole-3-acetic acid in each bacterial culture was analyzed by HPLC. Figure 7 ).

[0065] Example 7

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

[0067] Add a freeze-drying protectant (3% glycerol, 1% ascorbic acid, 5% mannitol, and 1% sodium dihydrogen phosphate) to the fermentation broth with the highest concentration of indole-3-acetic acid mentioned above, and mix thoroughly. Then, pre-freeze the treated fermentation broth at -80°C for 24 hours. After pre-freezing, perform formal freeze-drying of the fermentation broth using a vacuum freeze dryer at -90°C and 0.1 mbar for 72 hours, and grind it into powder. Figure 8 ).

[0068] Example 8

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

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

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

[0072] 2. SCAB_31831 gene replacement.

[0073] To confirm that the phenotype of the mutant strain ΔSCAB_31831 is entirely due to the SCAB_31831 gene mutation, this invention designed a SCAB_31831 gene complementation experiment for verification. pIB-31831 utilizes the strong promoter PermE* of the erythromycin resistance gene to initiate the transcriptional expression of the SCAB_31831 gene for complementation in the mutant strain ΔSCAB_31831. After fermentation culture and HPLC detection, the indole-3-acetic acid yield 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. The transcription of the indole-3-acetic acid biosynthesis gene was significantly increased after the deletion of the SCAB_31831 gene.

[0075] qRT-PCR results confirmed that, compared to the original strain 87.22, the expression levels of the indole-3-acetic acid biosynthesis genes iaaH and iaaM in the ΔSCAB_31831 mutant strain increased by 5.6-fold and 8.6-fold at 12 h, and by 1.9-fold and 1.7-fold at 24 h, respectively. Figure 6The results indicate that the deletion of the SCAB_31831 gene can significantly increase the transcriptional level of genes related to the biosynthesis of indole-3-acetic acid, thereby increasing the yield of indole-3-acetic acid.

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

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

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

[0079] This invention utilizes a freeze-drying protectant to enhance the stability of freeze-dried fermentation broth. The specific components are: 3% glycerol, 1% ascorbic acid, 5% mannitol, and 1% sodium dihydrogen phosphate. By thoroughly mixing the fermentation broth with this protectant and then freeze-drying it, the fermentation broth is transformed into a powder, which significantly enhances 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 within the protection scope of the present invention.

Claims

1. A method of treatment using *Streptomyces scabies* SCAB_31831 A method for increasing the yield of indole-3-acetic acid using genes, characterized in that, Through genetic engineering, Streptomyces scabies strain 87.22 was found to contain... SCAB_31831 Gene deletion was used to obtain a high-yield engineered strain of indole-3-acetic acid, which was then used to ferment and produce indole-3-acetic acid; among which, SCAB_31831 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application of the method as described in claim 1 in the preparation of a high-yield microbial preparation of indole-3-acetic acid.

3. A high-yield lyophilized bacterial powder for indole-3-acetic acid, characterized in that, First, through genetic engineering, the Streptomyces scabies strain 87.22 was modified... SCAB_31831 Gene deletion yielded a high-yield engineered strain of indole-3-acetic acid. SCAB_31831 The nucleotide sequence of the gene is shown in SEQ ID NO.1; then, 10 mM tryptophan was added to the fermentation medium of the high-yield engineered strain of indole-3-acetic acid, and the strain was fermented; finally, the obtained fermentation broth was freeze-dried to obtain bacterial powder.

4. The indole-3-acetic acid high-yield freeze-dried bacterial powder according to claim 3, characterized in that, The fermentation medium for the high-yield engineered strain of indole-3-acetic acid was oat bran medium.

5. The indole-3-acetic acid high-yield freeze-dried bacterial powder according to claim 3, characterized in that, After the fermentation of the high-yield engineered strain of indole-3-acetic acid is completed, a protective agent is added to the fermentation broth, followed by freeze-drying and grinding into powder.

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

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