Application of SCAB31801 gene or protein coded by SCAB31801 gene in regulation and control of synthesis of indole-3-acetic acid

Abstract

The invention discloses an application of an SCAB31801 gene or a protein coded by the SCAB31801 gene in regulation and control of synthesis of indole-3-acetic acid, and belongs to the technical field of genetic engineering. According to the invention, the SCAB31801 gene in streptomyces scabies is deleted through a genetic engineering way to obtain the indole-3-acetic acid high-yield strain, so that the yield of indole-3-acetic acid in streptomyces scabies is increased, and a technical support is provided for increasing the fermentation yield of indole-3-acetic acid in industrial production. When the SCAB31801 gene is deleted in streptomyces scabies, the yield of indole-3-acetic acid is increased by 1.3 times compared with that of an original strain; by adding 15mM tryptophan into the delta SCAB31801 mutant strain, the yield of the indole-3-acetic acid can be greatly increased by 33 times; the SCAB31801 gene is deleted and then the precursor tryptophan is added in a combined manner, so that the biosynthesis yield of the indole-3-acetic acid in streptomyces scabies can be directionally improved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly to the application of the SCAB_31801 gene or the protein encoded thereby in regulating the synthesis of indole-3-acetic acid. Background Art

[0002] Streptomyces is a common soil actinomycete that plays an important ecological role by decomposing organic matter and improving soil health. Its secondary metabolites are widely used in fields such as medicine and health, animal husbandry, industry, and agriculture. In Streptomyces, the synthesis of secondary metabolites is regulated in a complex and delicate manner, involving the coordinated action of multiple transcriptional regulatory factors, metabolic pathways, and gene regulatory networks. These transcriptional regulatory factors regulate the expression of secondary metabolic gene clusters through multi-dimensional regulatory mechanisms, thereby regulating the synthesis of secondary metabolites. By modifying the regulatory genes, the secondary metabolic gene clusters in Streptomyces can be effectively activated, thus increasing the yield of target secondary metabolites. The present invention aims to obtain Streptomyces strains with high-yield indole-3-acetic acid by genetically engineering the genes to produce indole-3-acetic acid.

[0003] Indole-3-acetic acid is an important plant hormone that regulates the growth rate of stems, inhibits lateral buds, and promotes root growth. In agricultural production, indole-3-acetic acid, as a growth regulator, can improve the yield and quality of crops. Therefore, increasing the yield of indole-3-acetic acid is of great significance for agricultural production. In the process of microbial synthesis of indole-3-acetic acid, tryptophan is the main precursor, which can be achieved through five different pathways. Among them, the tryptophan-dependent indole-3-acetamide (IAM) pathway is mainly used in Streptomyces scabies. In this pathway, tryptophan is catalyzed by tryptophan-2-monooxygenase to generate IAM, and then IAM is decomposed into indole-3-acetic acid under the action of indole-3-acetamide hydrolase.

[0004] The SCAB_31801 gene in Streptomyces scabies encodes a transcriptional regulatory factor of the AraC / XylS family, which is responsible for activating the transcriptional expression of the biosynthetic gene cluster of another secondary metabolite, thaxtomin A. The deletion of the SCAB_31801 gene can cause the strain to lose the ability to synthesize thaxtomin A. It is worth noting that the biosynthetic precursor of indole-3-acetic acid is also tryptophan, but the current research on the biosynthetic regulatory relationship between thaxtomin A and indole-3-acetic acid in Streptomyces scabies is still relatively limited. In addition, there is no relevant research on modifying the tryptophan metabolic pathway by genetic engineering means to increase the yield of indole-3-acetic acid in Streptomyces scabies. Summary of the Invention

[0005] The object of the present invention is to provide the application of the SCAB_31801 gene or the protein encoded thereby in regulating the synthesis of indole-3-acetic acid, so as to solve the problems existing in the above-mentioned prior art. The present invention discovers that there is a negative correlation between the SCAB_31801 gene of Streptomyces scabies and the biosynthesis of indole-3-acetic acid.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the application of the SCAB_31801 gene or the protein encoded thereby in regulating the synthesis of indole-3-acetic acid.

[0008] Another technical solution of the present invention is the application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the SCAB_31801 gene in enhancing the synthesis of indole-3-acetic acid.

[0009] A further technical solution of the present invention is the application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the SCAB_31801 gene in cultivating an engineered bacterium with high yield of indole-3-acetic acid.

[0010] A fourth technical solution of the present invention is an engineered bacterium with high yield of indole-3-acetic acid, and the engineered bacterium is a ΔSCAB_31801 deletion mutant of Streptomyces scabies 87.22.

[0011] A fifth technical solution of the present invention is a method for cultivating an engineered bacterium with high yield of indole-3-acetic acid. In the starting strain, the SCAB_31801 gene is silenced or knocked out, or the level of the protein encoded thereby is down-regulated to promote the synthesis of indole-3-acetic acid.

[0012] A sixth technical solution of the present invention is a method for producing indole-3-acetic acid by using the engineered bacterium, and 15 mM of tryptophan is added to the culture medium for fermentation culture.

[0013] Based on the above technical solutions, the present invention has the following technical effects:

[0014] The present invention identified the gene SCAB_31801 that has a negative correlation with indole-3-acetic acid biosynthesis. By deleting the SCAB_31801 gene in Streptomyces scabies through genetic engineering, a high-yield strain of indole-3-acetic acid was obtained, which improved the yield of indole-3-acetic acid in Streptomyces scabies, providing technical support for improving the fermentation yield of indole-3-acetic acid in industrial production. When the SCAB_31801 gene was deleted in Streptomyces scabies, the yield of indole-3-acetic acid increased by 1.3 times compared with the original strain; while adding 15 mM tryptophan to the ΔSCAB_31801 mutant strain could achieve a substantial 33-fold increase in the yield of indole-3-acetic acid; thus indicating that there is a negative correlation between the SCAB_31801 gene and indole-3-acetic acid biosynthesis, and the deletion of the SCAB_31801 gene combined with the addition of the precursor tryptophan can be used to directionally improve the biosynthesis yield of indole-3-acetic acid in Streptomyces scabies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 Schematic diagram of the construction process of the SCAB_31801 gene deletion mutant strain ΔSCAB_31801 in Example 6.

[0017] Figure 2 Double digestion verification diagram of the recombinant plasmid pUCTSR-Δ31801 used for SCAB_31801 gene knockout in Example 6. Among them, the upper band in 1-2 represents the pUCTSR-Δ31801-UP plasmid fragment of 5760 bp, and the lower band represents the downstream fragment of the SCAB_31801 gene of 1500 bp. M represents the 5000 bp DNA Marker.

[0018] Figure 3 PCR verification diagram of the mutant strain ΔSCAB_31801 in Example 6. Among them, 1 represents the positive control of 1475 bp, 2 represents the negative control of 951 bp, 3-4 represent the PCR amplification bands of the positive clones that are the same as 1, and M represents the 2000 bp DNA Marker.

[0019] Figure 4Verification diagram of double digestion of recombinant plasmid pIB-31801 for gene complementation of SCAB_31801 in Example 6. Among them, the upper band in lanes 1-2 represents the pIB139 plasmid fragment of 5900 bp, and the lower band represents the SCAB_31801 gene fragment of 951 bp. M represents the 5000 bp DNA Marker.

[0020] Figure 5 PCR verification diagram of the complemented strain ΔSCAB_31801 / pIB-31801 in Example 6. Among them, 1 represents the internal fragment of the acc(3)IV resistance gene on the pIB139 plasmid, i.e., the positive control of 750 bp, 2 represents the negative control, and the PCR amplification bands of 3-4 representing the screened positive clones are the same as 1. M represents the 2000 bp DNA Marker.

[0021] Figure 6 Analysis diagram of indole-3-acetic acid production of the ΔSCAB_31801 mutant strain in Example 6.

[0022] Figure 7 Transcription analysis diagram of genes related to indole-3-acetic acid biosynthesis in the ΔSCAB_31801 mutant strain in Example 6. Among them, it includes the indole-3-acetic acid biosynthesis genes iaaH (A) and iaaM (B).

[0023] Figure 8 Analysis diagram of indole-3-acetic acid production of the ΔSCAB_31801 mutant strain added with tryptophan in Example 6. Detailed implementation mode

[0024] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which will be obvious to those skilled in the art. Other embodiments obtained from the specification of this invention will be obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0028] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in this invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been publicly disclosed.

[0030] The embodiments of this invention provide the application of the SCAB_31801 gene or the protein encoded thereby in regulating the synthesis of indole-3-acetic acid.

[0031] In some specific embodiments, the nucleotide sequence of the SCAB_31801 gene is as shown in SEQ ID NO.1; the amino acid sequence of the protein encoded thereby is as shown in SEQ ID NO.2.

[0032] In some specific embodiments, overexpressing the SCAB_31801 gene or upregulating the level of the protein encoded thereby reduces the synthesis of indole-3-acetic acid; silencing or knocking out the SCAB_31801 gene or downregulating the level of the protein encoded thereby promotes the synthesis of indole-3-acetic acid.

[0033] The embodiments of this invention also provide the application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the SCAB_31801 gene in enhancing the synthesis of indole-3-acetic acid.

[0034] The embodiments of this invention also provide the application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the SCAB_31801 gene in cultivating an engineered bacterium with high yield of indole-3-acetic acid.

[0035] An embodiment of the present invention also provides a genetically engineered bacterium with high yield of indole-3-acetic acid, and the genetically engineered bacterium is a ΔSCAB_31801 deletion mutant of Streptomyces scabies 87.22.

[0036] An embodiment of the present invention also provides a method for cultivating a genetically engineered bacterium with high yield of indole-3-acetic acid. In the starting strain, the SCAB_31801 gene is silenced or knocked out, or the protein level encoded by it is down-regulated to promote the synthesis of indole-3-acetic acid.

[0037] In some specific embodiments, the starting strain includes Streptomyces scabies 87.22.

[0038] In some specific embodiments, a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of the SCAB_31801 gene is used to silence or knock out the SCAB_31801 gene and down-regulate the protein level encoded by it.

[0039] An embodiment of the present invention also provides a method for producing indole-3-acetic acid using the genetically engineered bacterium, and 15 mM tryptophan is added to the culture medium for fermentation culture.

[0040] In the present invention, the SCAB_31801 gene is deleted in Streptomyces scabies through genetic engineering to obtain a high-yield strain of indole-3-acetic acid in Streptomyces scabies, and then the obtained corresponding high-yield genetically engineered strain of indole-3-acetic acid in Streptomyces scabies is used for fermentative production of indole-3-acetic acid; wherein, the nucleotide sequence of the SCAB_31801 gene is as shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is as shown in SEQ ID NO.2.

[0041] In the step of deleting the SCAB_31801 gene in Streptomyces scabies, the specific Streptomyces scabies used is the Streptomyces scabies 87.22 strain, and the deletion of the gene is achieved through the suicide plasmid pUCTSR and homologous recombination technology. The SCAB_31801 gene encodes a transcriptional regulatory protein belonging to the AraC / XylS family. The expression product of the SCAB_31801 gene is used to inhibit the biosynthesis of indole-3-acetic acid.

[0042] The Streptomyces scabies 87.22 strain is a publicly available strain obtained by the public, provided by the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC 4.1765. The SCAB_31801 gene deletion mutant of Streptomyces scabies improves the synthesis of indole-3-acetic acid.

[0043] The strains and plasmids used in the embodiments of the present invention are shown in Table 1, and the primer sequences used are shown in Table 2.

[0044] Table 1 Strains and plasmids used in the examples of the present invention

[0045]

[0046] Table 2 Primers used in the examples of the present invention

[0047]

[0048] The Escherichia coli used in the examples of the present invention was cultured at 37 °C in liquid LB medium or on an LB solid plate supplemented with 2.0% agar. Streptomyces scabies 87.22 was cultured at 28 °C in TSB medium or on an SFM solid plate containing 2% agar. The medium used for the fermentation production of indole-3-acetic acid by Streptomyces scabies was OBB liquid fermentation medium and was cultured at 28 °C. The general operating techniques for Escherichia coli and Streptomyces scabies were in accordance with standard operations. The synthesis of primers and DNA sequencing were completed by General Biosystems (Anhui) Co., Ltd.

[0049] Example 1

[0050] The nucleotide sequence of the SCAB_31801 gene is shown as SEQ ID NO.1, and the protein sequence encoded thereby is shown as SEQ ID NO.2.

[0051] SEQ ID NO.1: GTGGCCTGTTCTGAACTGTTTGTCGCGCTCGGAGCCGAGAAGGGGGAAGCATCTTGCTTCACTCGATTTCCGGCCGACGCGCCGAGCGCAAGGTACTGCCTTTTGTCGAGCTCAGGCATGCAGATAAAATCTTTCAAGGCCGGCGGGGTCAAGGTGACGATCATAGATTCCGGTCCAGCCGTCATCGAGTTCGAGGCAATCAACTCGGAGGCGGCCTTGACGCCGCAGAGAACAGTCATATGCGTACTGTCAGGAATGGCGTTCATCGCTGGTACCGGAAACGGTACGGAGATCGACGCGGGGACGCTGGTTATGACGGACGGCGACGTTCCCTTTTCGATGAATGTGCCCGTTGCTTCGCGACTCCTCGTACTGCGTTTCGCCGACGAAGCGAAGGATGGACTCCCGGTGTCGCCTCGGGGGACTTTTATCGTGACGGATGCTGCCAAGGGTCCCGGATCCGGATTTCTTTTTTCGTTCTTGAATACCCTGGCTGTGGAGATGATGAAAACCGATGGGATTCTGTCCTCGTATATGGAGGAGGTCGTGCGCATCCTGGCGATCTCCGCGACGCGAATCGCATATGCCGAGCTCGGAAAGCATTACTCTGGGGGATGCGATCCACTTCTGATCGCGGTTCAGGAGTCGATCGACCGGCAGTTGGCCGACCCCGAGATCAGCCCGGCGACCCTCGCGGCCGAACACAACATATCGGTGCGTCAGTTACATCGAGTTTTCGGACCGATCGGGGAAAGCGTCATGAGCTATGTCAAACGCCGTCGCCTGGAGCGTTTCGCATGCGATCTGAGGGATCCGAGCCTGGGGCACCGGAAGATCAATGAGCTGGCGGCGGACTGGGGGATGCTGGATGCCGCGATGCTGAGCAGACACTTCCGCTGCGCCTACGGAATGTCGCCCCGCGATTACCGGAAGCAGCACTGTTTCACCTGA;

[0052] SEQ ID NO.2: VACSELFVALGAEKGEASCFTRFPADAPSARYCLLSSSGMQIKSFKAGGVKVTIIDSGPAVIEFEAINSEAALTPQRTVICVLSGMAFIAGTGNGTEIDAGTLVMTDGDVPFSMNVPVASRLLVLRFADEAKDGLPVSPRGTFIVTDAAKGPGSGFLFSFLNTLAVEMMKTDGILSSYMEEVVRILAISATRIAYAELGKHYSGGCDPLLIAVQESIDRQLADPEISPATLAAEHNISVRQLHRVFGPIGESVMSYVKRRRLERFACDLRDPSLGHRKINELAADWGMLDAAMLSRHFRCAYGMSPRDYRKQHCFT。

[0053] Construction of SCAB_31801 gene deletion mutant strain:

[0054] To construct the Streptomyces scabies SCAB_31801 gene deletion mutant strain, the internal sequence of the SCAB_31801 gene was replaced using the thiostrepton resistance gene tsr carried by the suicide plasmid pUCTSR ( Figure 1 ). Using the Streptomyces scabies genome as a template, the upstream and downstream homologous arm DNA fragments of 1500 bp each of the SCAB_31801 gene were amplified by PCR using 31801-UF / R and 31801-DF / R as primers respectively. The specific primer sequences are shown in Table 2. Among them, the underlined sequences of "AAGCTT", "TCTAGA", "GGATCC" and "GAATTC" are the cleavage sites of the restriction enzymes HindIII, XbaI, KpnI and EcoRI respectively.

[0055] The upstream and downstream DNA fragments were cloned into the pUCTSR plasmid using the above restriction enzymes to obtain the recombinant plasmid pUCTSR-Δ31801, and double digestion verification was carried out ( Figure 2 ). The recombinant plasmid pUCTSR-Δ31801 was transferred into Escherichia coli ET12567(pUZ8002), and pUCTSR-Δ31801 was introduced into Streptomyces scabies 87.22 by the intergeneric conjugation transfer technique. Thiostrepton was used to screen for resistant conjugants, and the selected strains were analyzed by PCR using the primers 31801-CF / R. The successfully verified positive strains were named ΔSCAB_31801 ( Figure 3 ). The specific primer sequences are shown in Table 2.

[0056] Example 2

[0057] Construction of the SCAB_31801 gene complementation strain:

[0058] To complement the SCAB_31801 gene in the ΔSCAB_31801 mutant strain, using the Streptomyces scabies genome as a template and 31801-CF / R as primers, a complete DNA fragment of the SCAB_31801 gene was amplified by PCR. The specific primer sequences are shown in Table 2. Among them, the underlined sequences of "GAATTC" and "AAGCTT" are the cleavage sites of restriction enzymes EcoRⅠ and HindⅢ, respectively.

[0059] The SCAB_31801 gene fragment was ligated to the plasmid pIB139 using the above restriction enzymes to construct the recombinant plasmid pIB-31801, and double digestion verification was carried out ( Figure 4 ). The recombinant plasmid pIB-31801 was transformed into ET12567(pUZ8002), and pIB-31801 was introduced into the deletion mutant strain ΔSCAB_31801 by conjugation transfer. Ampicillin was used to screen for resistant conjugants. After PCR verification, the complementation strain ΔSCAB_31801 / pIB-31801 was obtained ( Figure 5 ). Using the same method, the empty vector pIB139 was introduced into the mutant strain ΔSCAB_31801 to obtain the empty vector control strain ΔSCAB_31801 / pIB139 ( Figure 5 ).

[0060] Example 3

[0061] Detection of indole-3-acetic acid production in a series of Streptomyces scabies strains:

[0062] Spores of Streptomyces scabies 87.22 and the ΔSCAB_31801 series of strains with the same growth on the plate were inoculated into a TSB seed bottle and cultured by shaking at a rotation speed of 220 rpm and a temperature of 28 °C for 2 days. Subsequently, they were transferred to an OBB liquid medium and continued to be cultured by shaking at the same rotation speed and temperature for 7 days. After fermentation, indole-3-acetic acid in the fermentation broth was extracted and analyzed by HPLC, and its production was calculated using the indole-3-acetic acid standard curve ( Figure 6 ).

[0063] Example 4

[0064] Transcription analysis of related genes in ΔSCAB_31801:

[0065] Using an RNA extraction kit, RNA was extracted from Streptomyces scabies 87.22 and the mutant strain ΔSCAB_31801 during the fermentation culture process. After reverse-transcribing into cDNA, real-time fluorescence quantitative PCR was used to analyze the transcriptional levels of genes related to indole-3-acetic acid biosynthesis ( Figure 7 ).

[0066] Example 5

[0067] The ΔSCAB_31801 mutant strain combined with the precursor tryptophan to increase the indole-3-acetic acid yield:

[0068] Using the above method, the seed media of Streptomyces scabies 87.22 and the mutant strain ΔSCAB_31801 were obtained. Then, the seed media were respectively transferred to OBB liquid media containing different concentrations of tryptophan and cultured with shaking at 220 rpm and 28 °C for 7 days. After the fermentation ended, the indole-3-acetic acid yields in each bacterial liquid were analyzed by HPLC ( Figure 8 ).

[0069] The specific experimental results of the above examples:

[0070] 1. Basic information of the SCAB_31801 gene

[0071] The location of the SCAB_31801 gene on the chromosome of Streptomyces scabies can be found in NCBI. The nucleotide sequence of the SCAB_31801 gene is shown as SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown as SEQ ID NO.2.

[0072] 2. The indole-3-acetic acid yield increased significantly after the deletion of the SCAB_31801 gene

[0073] The construction process ( Figure 1 ) and PCR verification ( Figure 3 ) of the SCAB_31801 gene deletion mutant strain ΔSCAB_31801 are shown in the figure. ΔSCAB_31801 was fermented in OBB liquid medium for 7 days. After extraction and HPLC analysis, the indole-3-acetic acid yield of the mutant strain ΔSCAB_31801 was 1.3 times higher than that of the original strain 87.22, indicating a negative correlation between the SCAB_31801 gene and indole-3-acetic acid biosynthesis.

[0074] 3. Complementation of the SCAB_31801 gene

[0075] To confirm that the phenotype of the mutant strain ΔSCAB_31801 is completely caused by the mutation of the SCAB_31801 gene, the present invention designed a SCAB_31801 gene complementation experiment for verification. pIB-31801 uses the strong promoter PermE* of the erythromycin resistance gene to initiate the transcriptional expression of the SCAB_31801 gene and is used for the complementation of the mutant strain ΔSCAB_31801. After fermentation culture and HPLC detection, the indole-3-acetic acid production of the complemented strain ΔSCAB_31801 / pIB-31801 was restored to a level comparable to that of the original strain 87.22( Figure 6 ).

[0076] 4. Transcription of indole-3-acetic acid biosynthesis genes decreased significantly after deletion of the SCAB_31801 gene

[0077] qRT-PCR results confirmed that compared with the original strain 87.22, the expression levels of the indole-3-acetic acid biosynthesis genes iaaH and iaaM in the ΔSCAB_31801 mutant strain increased by 2.9-fold and 2.8-fold at 12 h; while at 24 h, they increased by 1.3-fold and 1.6-fold( Figure 7 ), indicating that the deletion of the SCAB_31801 gene can cause a significant increase in the transcriptional level of indole-3-acetic acid biosynthesis-related genes, thereby increasing the production of indole-3-acetic acid.

[0078] 5. Deletion of the SCAB_31801 gene combined with the precursor tryptophan further increases indole-3-acetic acid production

[0079] To further increase the production of indole-3-acetic acid, the present invention added tryptophan to the mutant strain ΔSCAB_31801 for fermentation culture. After HPLC analysis, compared with the original strain, when the tryptophan concentration was increased to 15 mM, the indole-3-acetic acid production of the mutant strain ΔSCAB_31801 increased by 33-fold( Figure 8 ), indicating that the strategy of deleting the SCAB_31801 gene combined with adding the precursor tryptophan can achieve a significant increase in indole-3-acetic acid production.

[0080] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of the SCAB_31801 gene or the protein encoded thereby in regulating indole-3-acetic acid synthesis.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the SCAB_31801 gene is as shown in SEQ ID NO.1; the amino acid sequence of the protein encoded thereby is as shown in SEQ ID NO.

2.

3. The application according to claim 1, wherein Overexpression of the SCAB_31801 gene or upregulation of the level of the protein encoded thereby reduces indole-3-acetic acid synthesis; silencing or knocking out the SCAB_31801 gene or downregulating the level of the protein encoded thereby promotes indole-3-acetic acid synthesis.

4. Use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the SCAB_31801 gene in enhancing indole-3-acetic acid synthesis.

5. Use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the SCAB_31801 gene in cultivating an engineered bacterium with high yield of indole-3-acetic acid.

6. An engineered bacterium with high yield of indole-3-acetic acid, characterized in that, The engineered bacterium is a ΔSCAB_31801 deletion mutant of Streptomyces scabies 87.

22.

7. A method for cultivating an engineered bacterium with high indole-3-acetic acid production, characterized in that, In the starting strain, silencing or knocking out the SCAB_31801 gene or downregulating the level of the protein encoded thereby promotes indole-3-acetic acid synthesis.

8. The method according to claim 7, characterized in that, The starting strain includes Streptomyces scabies 87.

22.

9. The method according to claim 7, wherein Using a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of the SCAB_31801 gene to silence or knock out the SCAB_31801 gene and downregulate the level of the protein encoded thereby.

10. A method for producing indole-3-acetic acid using the engineering bacteria described in claim 6, characterized in that, Fermentative culture is carried out by adding 15 mM of tryptophan to the medium.

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