Development and application of plant growth-promoting rhizobacteria staphylococcus pasteurii for producing 5-aminolevulinic acid

Abstract

The invention discloses development and application of plant growth-promoting rhizobacteria staphylococcus pasteurii capable of producing 5-aminolevulinic acid. The invention relates to the technical field of biology, in particular to development and application of plant growth-promoting rhizobacteria staphylococcus pasteurii for producing 5-aminolevulinic acid. According to the recombinant staphylococcus pasteurii disclosed by the invention, a hemB gene is knocked down; the recombinant staphylococcus pasteurii contains hemA, hemL2, pgr7 and ALAS (arabinose-L-aspartic acid), and also contains gltA, acs and pyk. The recombinant staphylococcus pasteurii SpALA as a high-yield PGPR of three growth-promoting factors has the advantages that the yield is increased by 26.99 times compared with that of an original strain ALA, meanwhile, siderophores and IAA can be secreted, the secretion amounts of the siderophores and IAA are increased by 13% and 6% respectively, the high-added-value compound ALA can be extracted from fermentation liquor of the recombinant staphylococcus pasteurii SpALA, the recombinant staphylococcus pasteurii SpALA can be directly applied to plant growth promotion and saline-alkali stress resistance, and the application range of the recombinant staphylococcus pasteurii SpALA is widened. And a more convenient and efficient application effect is presented.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the development and application of plant rhizosphere growth-promoting bacteria Staphylococcus pasteurianus that produces 5-aminolevulinic acid. Background Art

[0002] Plant growth promoting rhizobacteria (PGPR) and plant growth regulators (PGR) of biological origin are considered to be the most promising, green and efficient strategies to ensure food security, achieve green transformation of agriculture and build ecological civilization. PGPR replenishes iron for plants, enhances their stress resistance or directly promotes their growth by producing siderophores and indoleacetic acid (IAA). In addition to these classic growth promoting properties, new PGRs such as 5-aminolevulinic acid (ALA) have not been reported in PGPR related research. ALA is an essential precursor of tetrapyrrole compounds and is related to Mg. 2+ Chelation can form chlorophyll, and Fe 2+ Chelation forms heme, which plays a vital role in plant photosynthesis and respiration. ALA not only promotes crop growth and yield, enhances tolerance to various biotic and abiotic stresses, but also serves as a biodegradable, non-toxic herbicide and insecticide. With the development of green and sustainable agriculture, the demand for highly effective PGPR and novel PGR ALA is rapidly increasing across the agricultural sector.

[0003] Current research on PGPRs focuses on utilizing PGPRs with diverse functions to form microbial alliances, exogenous addition of PGRs to enhance the survival and growth-promoting effects of PGPRs, and various studies on PGPR-omics analysis. Current research on microbial production of ALA focuses on the use of model strains, Escherichia coli and Corynebacterium glutamicum. However, ALA produced by these model strains must be extracted and purified before agricultural application, which undoubtedly increases costs. Directly utilizing PGPRs to produce PGRALA and enhance their growth-promoting properties has yet to be reported. With advances in biotechnology and the need for weight loss and drug reduction in sustainable agricultural development, further development and utilization of PGPRs to produce PGRs is crucial. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to obtain a strain with high 5-aminolevulinic acid production, so as to increase the production of 5-aminolevulinic acid and enhance its growth-promoting properties.

[0005] In order to solve the technical problem of the present invention, the present invention provides a recombinant Staphylococcus pasteurianus, wherein the recombinant Staphylococcus pasteurianus knocks down the ALA dehydratase gene hemB; the recombinant Staphylococcus pasteurianus overexpresses the glutamyl tRNA reductase gene hemA, the glutamyl-1-semialdehyde aminotransferase gene heml2, the proton gradient regulation 7 gene pgr7 and the 5-aminolevulinic acid synthase gene ALAS, and also contains the citrate synthase gene gltA, the acetyl-CoA synthetase gene acs, and the pyruvate kinase gene pyk.

[0006] In the recombinant Staphylococcus pasteurianus described above, the hemA gene encodes any one of the following proteins:

[0007] A1) The amino acid sequence encoded by the hemA gene is the protein hemA with Genbank number WIF29717 (14 April 2023);

[0008] A2) a protein having hemA activity and having at least 80% identity to the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of A1);

[0009] A3) Connecting a tag to the N-terminus and / or C-terminus of A1) or A2) to obtain a fusion protein with hemA activity.

[0010] The heml2 gene encodes any of the following proteins:

[0011] B1) The amino acid sequence encoded by the heml2 gene is the protein heml2 with Genbank number WIF29719 (14 April 2023);

[0012] B2) a protein having heml2 activity and having at least 80% identity to the protein of B1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of B1);

[0013] B3) Connecting a tag to the N-terminus and / or C-terminus of B1) or B2) to obtain a fusion protein having heml2 activity.

[0014] The pgr7 gene encodes any of the following proteins:

[0015] C1) The pgr7 gene encodes the protein pgr7 whose amino acid sequence is sequence 2;

[0016] C2) a protein having pgr7 activity obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of C1) and having at least 80% identity with the protein of C1);

[0017] C3) Connecting a tag to the N-terminus and / or C-terminus of C1) or C2) to obtain a fusion protein having pgr7 activity.

[0018] The ALAS gene encodes any of the following proteins:

[0019] D1) ALAS gene from Rhodobacter sphaeroides 2.4.1 GeneID: 67448527, EC number: 2.3.1.37 protein ALAS;

[0020] D2) a protein having ALAS activity and having at least 80% identity to the protein of D1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of D1);

[0021] D3) Connecting a tag to the N-terminus and / or C-terminus of D1) or D2) to obtain a fusion protein with ALAS activity.

[0022] The gltA gene encodes any of the following proteins:

[0023] E1) The amino acid sequence of the gltA gene is the protein gltA with Genbank number WIF29721 (14 Apr 2023);

[0024] E2) a protein having gltA activity obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of E1) and having at least 80% identity with the protein of E1);

[0025] E3) Connecting a tag to the N-terminus and / or C-terminus of E1) or E2) to obtain a fusion protein having gltA activity.

[0026] The acs gene encodes any of the following proteins:

[0027] F1) The amino acid sequence encoded by the acs gene is the protein acs with Genbank number WIF29722 (14 Apr 2023);

[0028] F2) a protein having ACS activity and having at least 80% identity with the protein of F1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of F1);

[0029] F3) A fusion protein with ACS activity obtained by connecting a tag to the N-terminus and / or C-terminus of F1) or F2).

[0030] The pyk gene encodes any of the following proteins:

[0031] G1) The amino acid sequence encoded by the pyk gene is the protein pyk with Genbank number WIF29725 (14 Apr 2023);

[0032] G2) a protein having pyk activity obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of G1) and having at least 80% identity with the protein of G1);

[0033] G3) A fusion protein having pyk activity obtained by connecting a tag to the N-terminus and / or C-terminus of G1) or G2);

[0034] The hemB gene encodes any of the following proteins:

[0035] H1) The amino acid sequence encoded by the hemB gene is the protein hemB with Genbank number WIF29720 (14 Apr 2023);

[0036] H2) a protein having hemB activity, obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of H1) and having at least 80% identity with the protein of H1);

[0037] H3) A tag is connected to the N-terminus and / or C-terminus of H1) or H2) to obtain a fusion protein with hemB activity.

[0038] The present invention also provides a method for constructing the above-mentioned recombinant Staphylococcus pasteurianus.

[0039] The recombinant Staphylococcus pasteurianus described above was constructed according to the following method: the method includes knocking down the hemB gene described above in the recipient Staphylococcus pasteurianus, introducing the hemA, heML2, pgr7 and ALAS described above into the recipient Staphylococcus pasteurianus, and introducing the gltA, acs, and pyk described above into the recipient Staphylococcus pasteurianus.

[0040] The present invention also provides a method for producing 5-aminolevulinic acid, comprising culturing the recombinant Staphylococcus pasteurianus described above to obtain a fermentation product, and obtaining 5-aminolevulinic acid from the fermentation product.

[0041] The present invention also provides the above-mentioned method for producing 5-aminolevulinic acid or the use of the recombinant Staphylococcus pasteurianus in any of the following applications:

[0042] P1. Application in the production of 5-aminolevulinic acid;

[0043] P2. Application in increasing the yield of 5-aminolevulinic acid.

[0044] The present invention also provides the above-mentioned method for producing 5-aminolevulinic acid or the use of the recombinant Staphylococcus pasteurianus in any of the following applications:

[0045] (a1) promoting plant growth;

[0046] (a2) preparing products that promote plant growth;

[0047] (a3) IAA production;

[0048] (a4) preparing an IAA-producing product;

[0049] (a5) Siderophore production

[0050] (a6) preparing a siderophore-producing product;

[0051] (a7) Promote plant germination.

[0052] (a7) Improve the ability of plants to resist saline-alkali stress.

[0053] The plant growth promotion is embodied in whole or in part as follows:

[0054] (b1) promoting the increase of plant height under normal growth conditions or saline-alkali stress conditions;

[0055] (b2) promoting the increase of plant root length under normal growth conditions or saline-alkali stress conditions;

[0056] (b3) promoting the increase of plant biomass under normal growth conditions or saline-alkali stress conditions;

[0057] (b4) promoting an increase in plant dry weight under normal growth conditions or saline-alkali stress conditions;

[0058] (b5) promoting the increase of plant fresh weight under normal growth conditions or saline-alkali stress conditions.

[0059] The present invention also provides a method for promoting plant growth, comprising the following steps: applying the above-mentioned recombinant Staphylococcus pasteurianus to a test plant, thereby promoting plant growth.

[0060] The present invention enhances the key genes hemA, heml2, pgr7 and ALAS of the ALA synthesis module of Staphylococcus pasteurianus PK8 and knocks down the negative feedback gene hemB of ALA; at the same time, it enhances the key genes gltA, acs and pyk of the cell growth module of Staphylococcus pasteurianus PK8 and uses a temperature dynamic control strategy to balance the production of ALA and cell growth. Figure 1). The PGPR bacteria S. pasteuri SpALA fermentation broth developed by the present invention has the advantages of direct, simple and efficient application, eliminating the complicated processing and purification steps. In this way, the comprehensive growth-promoting and stress-resistant effects of the three growth-promoting factors (ALA, siderophore and IAA) secreted by the PGPR bacteria SpALA fermentation are directly utilized, and compared with the wild-type S. pasteuri PK8, the direct use of SpALA fermentation broth shows better growth-promoting and salt-alkali resistance, ensuring that the presence of PGPR bacteria SpALA exerts a sustainable growth-promoting and stress-resistant effect, and the application method is simpler, more effective and lower in cost, proposing a new strategy for the direct application of PGPR and its fermentation product ALA in sustainable agriculture.

[0061] Preservation Instructions

[0062] Species name: Staphylococcus pasteurianus

[0063] Latin name: Staphylococcus pasteuri

[0064] Strain ID: PK8

[0065] Depository: General Microbiology Center of China Culture Collection Administration

[0066] Abbreviation of depository institution: CGMCC

[0067] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0068] Deposit date: July 30, 2018

[0069] CGMCC registration number: CGMCC No.16178 BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a metabolic regulation diagram of ALA synthesis by Staphylococcus pasteurianus PK8. The green box represents the glycolysis pathway, the yellow box represents the byproduct acetic acid recycling pathway, and the blue box represents the TCA cycle pathway. These pathways are called upstream cell growth modules. The pink box represents the C5 pathway for ALA synthesis, the dark gray box represents the C4 pathway for ALA synthesis, and the light gray box represents the downstream metabolic pathway for ALA. These pathways are called ALA production modules. The solid line represents a single reaction, and the dotted line represents a series of reactions. The red color represents the genes of interest in this study. Intermediate product abbreviations: F6P, fructose-6-phosphate; F1,6P, fructose 1,6-diphosphate; PEP, phosphoenolpyruvate; AceCoA, acetyl-CoA; PYR, pyruvate; CIT, citrate; ICIT, isocitrate; α-KG, α-ketoglutarate; SucCoA, succinyl-CoA; Glu, glutamate; Gln tRNA, glutamyl-tRNA; GSA, glutamate 1-semialdehyde aminotransferase; PBG, porphobilinogen. Gene abbreviations: pfkA, phosphofructokinase gene; pyk, pyruvate kinase gene; acs, acetyl-CoA synthetase gene; gltA, citrate synthase gene; icd, isocitrate dehydrogenase gene; hemA, glutamyl-tRNA reductase gene; pgr7, hemA kinase gene; heml1, glutamyl-1-semialdehyde aminotransferase gene 1; heml2, glutamyl-1-semialdehyde aminotransferase gene 2; ALAS, ALA synthetase gene; hemB, ALA dehydratase gene.

[0071] Figure 2 The ALA production and growth conditions of the SpALA strain and its construction process.

[0072] Figure 3 The transcription levels of hemB, hemA, heml1 and heml2 genes in S. pasteuri PK8 and PKB were determined by RT-qPCR, respectively.

[0073] Figure 4 This is the fermentation curve of the SpALA strain expanded culture in a fermenter.

[0074] Figure 5 Figure 5. Siderophore and IAA production of S. pasteuri PK8 and SpALA, and their effects on maize germination. a. Siderophore and IAA production; b. Effect on maize germination.

[0075] Figure 6 Figure 3. Effects of three SpALA treatments on maize seedling growth and stress resistance compared to S. pasteuri PK8. a. SpALA growth-promoting and salt-alkali-resistant effects; b. SpALA growth-promoting effects on maize plant height; c. SpALA growth-promoting effects on maize root length. Con: control treated with equal amounts of sterile water; 5ALA: ALA purified from SpALA fermentation diluted to 5 mg / L with sterile water; Bacteria: ALA purified from SpALA fermentation diluted to 10 mg / L with sterile water. 8 CFU / mL of bacteria; Supernatant: Filter-sterilized fermentation broth supernatant diluted with sterile water to 5 mg / L ALA; Fermentation broth: Fermentation broth directly diluted to 5 mg / L ALA; Na8Con: Control under saline-alkali stress; Na8PK8 fermentation broth: PK8 fermentation broth directly diluted to 5 mg / L ALA-treated seeds sown in a 1 / 2 MS under saline-alkali stress; Na8ConSpALA fermentation broth: SpALA fermentation broth directly diluted to 5 mg / L ALA-treated seeds sown in a 1 / 2 MS under saline-alkali stress. Different letters in the bar graph indicate significant differences between the data (Duncan test, p < 0.05). DETAILED DESCRIPTION

[0076] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0077] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0078] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0079] In the following examples, DNA Maker, bacterial genomic DNA extraction kit, rapid plasmid mini-extraction kit (centrifugal column type) and ordinary agarose gel DNA recovery kit were all purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the Tap enzyme used in PCR was purchased from Beijing Polymer Biotechnology Co., Ltd., the high-fidelity enzyme Tks Gflex DNA Polymerase was purchased from Takara; and restriction endonucleases were purchased from Takara.

[0080] In the following examples, Staphylococcus pasteuri PK8 is a high-ALA-producing PGPR strain isolated from the rhizosphere soil of Portulaca oleracea. It was collected on July 30, 2018 at the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the registration number of the collection center is CGMCC No. 16178.

[0081] In the following examples, Staphylococcus aureus RN4220 was donated by Professor Sun Baolin of the University of Science and Technology of China and has been recorded in: Habib, G., Zhu, J., and Sun, B. (2020). A novel type I toxin-antitoxin system modulates persister cell formation in Staphylococcus aureus. Int J Med Microbiol 310, 151400. The public can obtain this biological material from the applicant for use only in repeating the experiments of the present invention and cannot be used for other purposes.

[0082] In the following examples, the vector pRMC2 was donated by Professor Sun Baolin of the University of Science and Technology of China and has been described in: Habib, G., Zhu, J., and Sun, B. (2020). A novel type I toxin-antitoxin system modulates persister cell formation in Staphylococcus aureus. Int J Med Microbiol 310, 151400. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0083] In the following examples, the vector pnCasSA-BEC was a gift from Professor Quanjiang Ji of ShanghaiTech University and has been described in: Gu, T., Zhao, S., Pi, Y., Chen, W., Chen, C., Liu, Q., Li, M., Han, D., and Ji, Q. (2018). Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase. Chem. Sci. 9, 3248-3253. The public can obtain this biological material from the applicant for use only in repeating the experiments of the present invention and cannot be used for other purposes.

[0084] The data were processed using IBM SPSS Statistics 25 statistical software in the following examples. The experimental results are expressed as mean ± standard deviation, and the Duncan test was used. p < 0.05 indicated a significant difference, or the t-test was used: p < 0.05, * indicated a significant difference, p < 0.01, ** indicated a very significant difference, p < 0.001, *** indicated an extremely significant difference.

[0085] In the following examples, MKB iron-restricted medium: (A) 50 g casamino acids, 15 mL glycerol, 785 mL deionized water; (B) 2.5 g K₂HPO₄ dissolved in 100 mL; (C) 2.5 g MgSO₄·7H₂O dissolved in 100 mL. After preparation, sterilize each medium separately at 115°C for 20 min, pH 7.2, and mix thoroughly before use.

[0086] In the following examples, fermentation medium LBGPM was prepared as follows (1 L): 50 g glucose, 10 g tryptone, 10 g yeast extract, 6 g rice syrup, 2.5 g NaCl, 1.0 g MgSO4·7H2O, 17.1 g Na2HPO4·12H2O, 3 g KH2PO4, with the balance being water, pH 7.0. Glucose, Na2HPO4·12H2O, KH2PO4, and MgSO4·7H2O were sterilized separately at 115°C for 20 min.

[0087] In the following examples, the specific preparation method of sodium acetate buffer at pH 4.6 can be as follows: measure 5.7 mL of glacial acetic acid, weigh 8.2 g of anhydrous sodium acetate, add water to make up to 100 mL, and store at 4°C for use.

[0088] In the following examples, the specific preparation method of Ehrlich's reagent can be as follows: weigh 1g of p-dimethylaminobenzaldehyde, add 30mL of glacial acetic acid, then add 8mL of perchloric acid (70%), dilute to 50mL with glacial acetic acid, and use it immediately or store it at 4°C.

[0089] In the following examples, the CAS assay solution includes the following: (A) 1 mM ferric chloride stock solution: 0.2703 g ferric chloride hexahydrate diluted in 1 L of 10 mM hydrochloric acid; (B) CAS stock solution: 0.2421 g chrome azurol (CAS) diluted in 200 mL of deionized water; (C) hexadecyltrimethylammonium bromide (HTDMA) solution: 0.0219 g HTDMA diluted in 50 mL of deionized water; and (D) piperazine buffer: 4.3079 g anhydrous piperazine aqueous solution adjusted to pH 5.6 with hydrochloric acid and diluted in 30 mL of deionized water. After each solution was prepared, 1.5 mL of solution A was added to 7.5 mL of solution B, mixed thoroughly, and 50 mL of solution C was added while stirring, followed by 30 mL of solution D. Finally, the volume was adjusted to 100 mL with deionized water.

[0090] In the following examples, Salkowski reagent: weigh 1.8 g FeCl3, add 64.4 mL concentrated sulfuric acid, and then dilute to 150 mL with deionized water.

[0091] In the following examples, primers were designed according to the seamless cloning principle, and primer synthesis and sequencing were completed by Sangon Gene Co., Ltd. The specific sequences are shown in Table 1 below.

[0092] Table 1. Primers used in the present invention

[0093]

[0094]

[0095] Table 2. Strains and plasmids used in the present invention

[0096]

[0097] The experimental methods in the following examples are conventional methods unless otherwise specified. The detailed steps of some of the experimental methods in the following examples are as follows:

[0098] A. ALA determination: ALA was determined using the Ehrlich's reagent colorimetric method. Staphylococcus pasteurianus fermentation broth was centrifuged at 12,000 × g for 1 minute. The supernatant was diluted to an appropriate concentration. 400 μL of the supernatant was added to 200 μL of sodium acetate buffer and 100 μL of acetylacetone. After boiling in a water bath for 15 minutes and cooling to room temperature, 700 μL of Ehrlich's reagent was added. Mix thoroughly and react in the dark at room temperature for 20 minutes. OD was measured. 554 , with the corresponding unfermented culture medium as the control. Note that the colorimetry should be completed within half an hour.

[0099] B. Determination of siderophore production: Culture the bacteria in MKB iron-limited medium, and then use the classic chrome azurol (CAS) method to determine the siderophore production. Centrifuge the Staphylococcus pasteurianus fermentation broth at 12,000 × g for 1 minute, take an appropriate amount of the supernatant, add CAS detection solution in a 1:1 ratio, let it react for 2 hours, and measure the OD 630 , the value obtained is A; the same treatment is performed with the corresponding culture medium, and the value obtained is Ar. The calculation formula for the relative content of siderophores (SU) is: SU = 1-A / Ar.

[0100] C. IAA determination: The bacterial culture was cultured in a medium containing 100 mg / L tryptophan and then determined using the classic PC colorimetric method. The Staphylococcus pasteurianus fermentation broth was centrifuged at 12,000 × g for 1 min, mixed with Salkowski reagent in a 1:1 ratio, and then reacted in the dark at room temperature for 30 min. A 1:1 mixture of Salkowski and deionized water was used as a control and the OD was measured. 530 .

[0101] D. In the following examples, the culture method of Staphylococcus pasteurianus PK8 is as follows:

[0102] Remove Staphylococcus pasteurii PK8 from -80°C storage and streak using a sterile inoculating loop onto fresh TSB solid medium (17 g tryptone, 3 g phytone, 5 g sodium chloride, 2.5 g potassium dihydrogen phosphate, 2.5 g glucose, adjusted to pH 7.0) for activation. Select a single freshly grown S. pasteurii PK8 colony from the streaked plate and inoculate it into a test tube containing 4 mL of TSB for activation overnight at 37°C and 200 rpm. Transfer the inoculum to a shake flask at 1% of the volume and continue incubation at 37°C and 200 rpm for 24 hours to obtain a Staphylococcus pasteurii PK8 culture suspension.

[0103] E. In the following examples, extraction, PCR amplification and plasmid construction of Staphylococcus pasteurianus PK8 genome:

[0104] An appropriate amount of bacterial slurry to be tested was collected, 300 μL of 40 U / mL lysostaphin was added, and the mixture was shaken at 37°C and 200 rpm for 45 min, followed by centrifugation to remove the supernatant. The target genome was then extracted using a common bacterial genome extraction kit.

[0105] PCR amplification and plasmid construction were performed according to conventional methods. The specific steps are as follows:

[0106] (1) PCR amplification of target gene

[0107] Tks Gflex PCR amplification system and reaction

[0108] <![CDATA[2×Gflex PCR Buffer(Mg 2+ ,dNTP plus)]]> 25 μL Primer F (10 μM) 1 μL Primer R (10μM) 1 μL DNA template <500ng Tks Gflex DNA Polymerase(1.25units / μL) 1 μL <![CDATA[ddH2O]]> XμL Overall system 50μL

[0109] Reaction procedure: pre-denaturation at 94°C for 1 min; 35 cycles at 98°C for 10 s, Tm-5°C for 15 s, and 68°C for 30 s / kb; and extension at 68°C for 5 min.

[0110] (2) Electrophoresis, recovery and purification of PCR products

[0111] Purify and recover each gene fragment according to the instructions of the universal DNA purification and recovery kit.

[0112] (3) Enzyme digestion of plasmid

[0113] 10×M buffer 10 μL pRMC2 xμL KpN 5μL EcoRI 5μL <![CDATA[ddH2O]]> yμL Overall system 100 μL

[0114] Incubate the enzyme digestion reaction in a metal bath at 37°C for 2-4 hours.

[0115] (4) Connection

[0116] The plasmid fragment and gene fragment were measured by NanoPhotometer ultramicro spectrophotometer and connected at a molar ratio of 1:3 or 1:7 using the EZ-S one-step seamless cloning kit.

[0117] The seamless cloning connection system is as follows:

[0118] 2×OneStep Cloning Mix 5μL Plasmid fragment xμL gene fragments yμL <![CDATA[ddH2O]]> zμL Overall system 10 μL

[0119] React in a PCR instrument at 50°C for 20 min.

[0120] Finally, colony PCR and sequencing were used to verify the correctness of the gene and plasmid construction. The correctly constructed plasmid was selected and transformed into the competent intermediate host Staphylococcus aureus RN4220 by electroporation, and the modified plasmid was then extracted from the RN4220 strain.

[0121] F. Construction of engineered strains

[0122] 1. Preparation of competent cells of S. pasteurii PK8

[0123] (1) Take out the S. pasteurii PK8 strain stored at -80°C, streak it onto a TSB solid plate with a sterile inoculating loop, and incubate it at 37°C overnight;

[0124] (2) Pick a single colony of freshly grown S. pasteurii PK8 from the streaked plate and transfer it to TSB liquid medium and culture at 37°C and 200 rpm until the logarithmic phase;

[0125] (3) Inoculate 1% of the inoculum into freshly prepared and filter-sterilized TSB liquid medium and culture at 37°C and 200 rpm until the OD 600 At this time, different types and concentrations of cell wall weakening agents are added according to needs (the final optimized condition is to add penicillin with a final concentration of 15 μg / mL and 0.01% Tween-80), and the culture is continued until the OD 600 About 0.40;

[0126] (4) Aseptically dispense the bacterial suspension into pre-chilled 100 mL centrifuge tubes, 50 mL per tube, and place on ice for 10 min to stop growth.

[0127] (5) After balancing the centrifuge tube, centrifuge at 5000 × g and 4°C for 15 min to collect the cells (the centrifuge must be pre-cooled in advance);

[0128] (6) Discard the supernatant as much as possible (the centrifuge tube can be inverted on sterile absorbent paper to remove as much residual culture medium as possible), gently resuspend the cells in 1-2 mL of pre-chilled 0.5 M sucrose, and then make up the volume to 50 mL. Gently invert to mix, and let it stand on ice for 15 min.

[0129] (7) Collect the cells by centrifugation at 5000 × g and 4°C for 15 min, discard the supernatant, and gently resuspend the cells in 1-2 mL of pre-chilled 0.5 M sucrose to make up the volume to 25 mL. Gently invert to mix, and let stand on ice for 30 min.

[0130] (8) Centrifuge again at 5000 × g, 4°C for 15 min to collect the cells, discard the supernatant, and gently resuspend in pre-chilled 0.5 M sucrose.

[0131] (9) Use a pre-cooled blue pipette tip to quickly dispense the cells into pre-cooled 1.5 mL centrifuge tubes (100 μL / tube) for immediate use.

[0132] 2. Electroporation of S. pasteurii PK8

[0133] (1) Place the plasmid extracted from the intermediate host RN4220, the electroporation cup, and the filter-sterilized TSB on ice for precooling;

[0134] (2) Use a pipette to transfer 10 μL of plasmid into 100 μL of competent cells and place on ice for 30 min;

[0135] (3) Set the voltage of the electroporator to 2.0-2.5 kV (finally select 2.5 kV), transfer the competent cells with the plasmid into the electrode cup, and quickly electroporate with the electroporator;

[0136] (4) After electroporation, 600 μL of TSB medium was quickly added, and the cells were allowed to stand on ice for 5 min before the bacterial solution was transferred from the electroporation cup to a centrifuge tube. The cells were cultured at 30 or 37°C and 200 rpm for 2 h for recovery.

[0137] (5) After rejuvenation, spread the plate on a TSB solid plate of the corresponding resistance and culture it upside down at a constant temperature of 30 or 37°C until single colonies of appropriate size appear.

[0138] G. Verification of engineered Staphylococcus pasteurii strains

[0139] To detect the engineered strain of S. pasteuri using PCR, pick a fresh single colony in a clean bench and add it to 20 μL of sterile water. Mix well, then boil in water for 5 minutes, immediately ice bath for 4 minutes; then boil in water for 3 minutes, immediately ice bath for 2 minutes; then boil in water for 3 minutes, immediately ice bath for 2 minutes. Centrifuge at 10,000 × g for 5 minutes, then take the supernatant as a template and perform PCR according to the following PCR reaction system. The reaction system is as shown in the table below:

[0140]

[0141]

[0142] The forward and reverse primers in the reaction system are shown in Table 1. The reaction procedure was as follows: initial denaturation at 95°C for 3 min; 30 cycles at 94°C for 25 s, 50-72°C for 25 s, and 72°C for 30 s / kb; and extension at 72°C for 10 min.

[0143] For engineered strains that down-regulate hemB using CRISPER, PCR is followed by sequencing verification.

[0144] H. Elimination of endogenous plasmids in Staphylococcus pasteurianus PK8

[0145] S. pasteuri PK8 was first cultured in TSB at 37°C for 12 hours. A 1% inoculum was then transferred to TSB containing 0.02% SDS and cultured at 42°C for an additional 12 hours. Appropriate dilutions of the cells were plated onto TSB plates. Colony PCR was performed to detect the presence of the Erm resistance gene in individual colonies on the plates, confirming the elimination of the endogenous plasmid. This was further validated by the sensitivity of strains that had undergone endogenous plasmid elimination to Erm resistance. Strains confirmed by colony PCR were streaked onto TSB plates containing 5 μg / mL Erm and onto TSB plates without resistance. If the strain failed to grow on the resistant plate but did grow on the non-resistant plate, the endogenous plasmid was successfully eliminated.

[0146] I. Fermentation culture of S. pasteur engineered strain

[0147] Shake flask culture of engineered S. pasteuri strains: A single fresh colony was inoculated into TSB medium containing 5 μg / mL Cm (chloramphenicol) and / or 5 μg / mL Erm (erythromycin). Culture was continued at 37°C or 30°C at 200 rpm until late logarithmic phase. A 1% inoculum was then inoculated into a 150 mL Erlenmeyer flask containing 30 mL of LBGP fermentation medium. Culture was continued at 37°C or 30°C at 200 rpm for 24 hours. Gene expression was induced with ATC at a final concentration of 320 ng / mL. Samples were collected 24 hours after culture for measurement of cell biomass and 5-aminolevulinic acid production.

[0148] Fermenter culture of the engineered S. pasteuri strain: A single fresh colony was inoculated into TSB medium containing 5 μg / mL Cm and 5 μg / mL Erm. Culture was continued at 30°C, 200 rpm, until late logarithmic phase. A 10% inoculum was then added to a 5-L fermenter containing 3.5 L of LBGPM fermentation medium. Culture was continued at 30°C for the first 12 hours, maintaining the pH at approximately 6.8 with aqueous ammonia. The fermenter was then incubated at 37°C for the next 12 hours, with the pH maintained at a natural pH. Samples were collected at 24 hours for measurement of bacterial biomass and 5-aminolevulinic acid production.

[0149] Example 1: Construction of a recombinant Staphylococcus pasteurianus strain SpALA that produces high 5-aminolevulinic acid

[0150] 1. Construction of recombinant Staphylococcus pasteurianus strain RA

[0151] The inducible plasmid pRMC2 was double-digested with KpnI and EcoRI to obtain the pRMC2 plasmid fragment. The hemA gene (GeneID: OQ828633, April 14, 2023) was PCR-amplified from DNA of S. pastoris PK8 using RA-F / R primers. The pRMC2 plasmid fragment was ligated with the hemA gene fragment using seamless cloning and then transformed into competent E. coli DH5a cells to obtain recombinant E. coli containing the pRA plasmid. The pRA plasmid was extracted from the recombinant E. coli and purified for later use.

[0152] PCR and sequencing confirmed that the pRA plasmid was a vector for overexpressing the hemA gene, which was seamlessly cloned and connected between the restriction endonuclease KpnI recognition site and the EcoRI recognition site of the pRMC2 plasmid.

[0153] The pRA plasmid was transformed into S. aureus RN4220 to obtain the positive RN4220 strain RN4220 / pRA. Plasmid pRA was then extracted from the positive RN4220 / pRA and transformed into the starting strain S. pasteuri PK8. The resulting S. pasteuri strain containing the pRA plasmid was designated RA. S. pasteuri PK8 containing the empty pRMC2 plasmid served as a control (abbreviated as SpR).

[0154] The recombinant strain RA and the control strains PK8 and SpR were fermented using the method described above (I. Fermentation culture of S. pasteur engineered strains). Figure 2 As shown, compared with SpR, overexpression of the hemA gene increased the ALA production to 309.48 mg / L, which increased the ALA production by 144%.

[0155] 2. Construction of recombinant Staphylococcus pasteurianus strain RA2

[0156] The hemA and heml2 genes were co-expressed to obtain strain RA2. The specific steps for constructing strain RA2 are as follows:

[0157] The inducible plasmid pRMC2 was double-digested with KpnI and EcoRI to obtain the pRMC2 plasmid fragment. The hemA and heml2 genes were PCR amplified from DNA of S. pastoris PK8 using primers RA-F / R and RL2-F / R, respectively. The resulting hemA gene (GeneID: OQ828633, 14 April 2023) and heml2 gene (GeneID: OQ828635, 14 April 2023) were simultaneously ligated using seamless cloning. The pRMC2 plasmid fragment, hemA, and heml2 gene fragments were then transformed into competent E. coli DH5a cells to obtain recombinant E. coli / pRA2 containing the pRA2 plasmid. The pRA2 plasmid was then isolated and purified for later use.

[0158] PCR and sequencing verified that the correct pRA2 plasmid was constructed by seamlessly ligating the hemA and heML2 genes between the KpnI and EcoRI recognition sites of the pRMC2 plasmid, resulting in an overexpression vector for the hemA and heML2 genes. This pRA2 plasmid was transformed into S. aureus RN4220, yielding the positive RN4220 strain RN4220 / pRA2. The plasmid pRA2 was then extracted and transformed into the starting strain S. pasteurii PK8. The resulting S. pasteurii PK8 strain containing the pRA2 plasmid was designated RA2. A S. pasteurii PK8 strain containing an empty pRMC2 plasmid served as a control (abbreviated as SpR).

[0159] The recombinant strain RA2 obtained above was fermented using the method described above (fermentation culture of I. S. pasteur engineered strain). Figure 2 As shown: Induced overexpression of hemA and heml2 genes increased the ALA production to 494.27 mg / L, which was further increased by 60% compared with the RA strain.

[0160] 3. Construction of recombinant Staphylococcus pasteurianus strain RA72

[0161] The hemA, pgr7, and heml2 genes were co-expressed to obtain strain RA72. The specific steps for constructing strain RA72 are as follows:

[0162] The inducible plasmid pRMC2 was double-digested with KpnI and EcoRI to obtain the pRMC2 plasmid fragment. The hemA and heml2 genes were PCR amplified from DNA of S. pasteurii PK8 using primers RA-F / R and RL2-F / R, respectively. The resulting hemA gene (GeneID: OQ828633, 14 April 2023) and heml2 gene (GeneID: OQ828635, 14 April 2023) were amplified. The codon-optimized pgr7 gene from Arabidopsis thaliana (nucleotide sequence: Sequence 1 in the sequence listing) was used. The pRMC2 plasmid fragment, hemA, pgr7 and heml2 gene fragments were simultaneously connected by seamless cloning, and then transformed into E.coli DH5a competent cells to obtain recombinant E.coli E.coli / pRA72 containing the pRA72 plasmid. The pRA72 plasmid in the recombinant E.coli was extracted and purified for later use.

[0163] The pRA72 plasmid, verified by PCR and sequencing, was constructed by seamlessly ligating the hemA, pgr7, and heML2 genes between the KpnI and EcoRI recognition sites of the pRMC2 plasmid, resulting in an overexpression vector for the hemA, pgr7, and heML2 genes. This pRA72 plasmid was transformed into S. aureus RN4220, yielding the positive RN4220 strain RN4220 / pRA72. This plasmid was then extracted and transformed into the starting strain S. pasteurii PK8. The resulting S. pasteurii PK8 strain harboring the pRA72 plasmid was designated RA72.

[0164] The recombinant strain RA72 obtained above was fermented using the method described above (I. Fermentation culture of S. pasteur engineered strain). Figure 2 As shown: Induced overexpression of hemA, pgr7 and heml2 genes increased the ALA production to 619.52 mg / L, which was further increased by 25% compared with the RA2 strain.

[0165] 4. Construction of recombinant Staphylococcus pasteurianus strain RA72S

[0166] The hemA, pgr7, heml2, and ALAS genes were co-expressed to obtain strain RA72S. The specific steps for constructing strain RA72S are as follows:

[0167] The inducible plasmid pRMC2 was double-digested with KpnI and EcoRI to obtain the pRMC2 plasmid fragment. The co-expressed gene fragments hemA, pgr7, and heml2 were PCR amplified from the pRA72 plasmid using primers RA-F and RL2-R, respectively. The ALAS gene sequence (GeneID: 67448527, EC number: 2.3.1.37) from R. sphaeroides 2.4.1 was obtained from Biotechnology Information Services (Bioinformatics). The pRMC2 plasmid fragments were simultaneously ligated using seamless cloning, followed by the hemA, pgr7, heml2, and ALAS gene fragments. Subsequently, the fragments were transformed into competent E. coli DH5a cells to obtain recombinant E. coli / pRA72S containing the pRA72S plasmid. The pRA72S plasmid was then isolated and purified for later use.

[0168] The pRA72S plasmid, verified by PCR and sequencing, was constructed by seamlessly ligating the hemA, pgr7, heml2, and ALAS genes between the KpnI and EcoRI recognition sites of the pRMC2 plasmid, resulting in an overexpression vector for the hemA, pgr7, heml2, and ALAS genes. This pRA72S plasmid was transformed into S. aureus RN4220, yielding the positive RN4220 strain RN4220 / pRA72S. The plasmid pRA72S was then extracted and transformed into the starting strain S. pasteurii PK8. The resulting S. pasteurii PK8 strain harboring the pRA72S plasmid was designated RA72S.

[0169] The recombinant strain RA72S obtained above was fermented using the method described above (I. Fermentation culture of S. pasteur engineered strain). Figure 2 As shown: Induced overexpression of hemA, pgr7, heml2 and ALAS genes increased the ALA production to 967.13 mg / L, which was further increased by 56% compared with the RA72 strain.

[0170] 5. Construction of recombinant Staphylococcus pasteurianus strain BRA72S

[0171] To prevent negative feedback from the ALA production module, this study used the CRISPR system to create a nonsense mutation (terminator) in the hemB gene of S. pasteuri PK8 (GeneID: OQ828636, 14 April 2023), generating a PKB mutant. Based on the pnCasSA-BEC mutagenesis principle (Gu et al., 2018), the glutamine Gln(Q) (CAA) at position 177 of the hemB gene of S. pasteuri PK8 was mutated to a stop codon (TAA), prematurely terminating transcription and achieving hemB downregulation. Specific construction steps were followed as described in the literature (Gu et al., 2018).

[0172] (1) Design of targeting primers: Select a 20 bp spacer sequence before the PAM site (NGG) of the hemB gene and design BF / R;

[0173] (2) Phosphorylation: Reaction system (50 μl): BF (100 μM) 1 μl, BR (100 μM) 1 μl, 10× T4 DNA ligase buffer (NEB) 5 μl, T4 polynucleotide kinase (Takara) 1 μl, ddH2O 42 μl. Incubate at 37°C in a metal bath for 1 h.

[0174] (3) Annealing: Mix 0.5 μL of 5M NaCl with the phosphorylated product. Incubate at 95°C for 3 min in a PCR instrument.

[0175] Then, the solution was slowly cooled to room temperature to anneal the BF / BR, and the annealed product was diluted 10 times with ddH2O.

[0176] (4) Golden gate assembly: Golden gate reaction system (10 μL): pnCasSA-BEC plasmid (20 fmol / μL) 1 μL, diluted annealed oligos (100 fmol) 1 μL, 10x T4 DNA ligase buffer (NEB) 1 μL, T4 DNA ligase (NEB) 0.5 μL, BsaI (NEB) 0.5 μL, ddH2O 6 μL. PCR reaction program: 37°C for 3 min, 16°C for 4 min, 25 cycles; 50°C for 5 min; 80°C for 15 min; incubation at 10°C.

[0177] (4) Transformation: The above product was transformed into 100 μL E. coli DH5α competent cells, and the successful construction of the pB plasmid was confirmed by PCR and sequencing. The pB plasmid was then electroporated into RN4220 competent cells, and the pB plasmid was extracted from it and electroporated into S. pasteurii PK8 competent cells. DNA of the transformants was extracted, and the correct mutant strain was verified by PCR sequencing. The strain was then cultured in TSB medium containing 5 μg / mL Cm at 30°C overnight, inoculated into TSB medium without Cm at a 0.1% inoculum, and cultured at 42°C for 12 hours. The culture was spread onto TSB plates without Cm and cultured at 37°C overnight. Single colonies were streaked on TSB plates and TSB + 5 μg / mL Cm plates. Those that grew normally on TSB but could not grow on TSB + 5 μg / mL Cm plates were PKB mutants.

[0178] The transcription levels of hemB, hemA, heml1 and heml2 genes in S. pasteuri PK8 and PKB were determined by RT-qPCR. Figure 3 The hemB transcription level of the PKB strain was significantly reduced to 62% of the control. Finally, the present invention obtained a mutant strain with successful hemB downregulation, named PKB.

[0179] The pRA72S plasmid was electroporated into the PKB strain to obtain a strain with down-regulated hemB and over-expressed hemA, pgr7, heml2 and ALAS genes, which was named BRA72S strain.

[0180] The recombinant strain RA72S obtained above was fermented using the method described above (I. Fermentation culture of S. pasteur engineered strain). Figure 2 As shown: Downregulation of hemB and simultaneous overexpression of hemA, pgr7, heml2, and ALAS genes resulted in ALA production reaching 1389.14 mg / L, a further 44% increase compared to the RA72S strain. However, the biomass of the BRA72S strain decreased significantly by 25% compared to the wild-type S. pasteuri PK8, a phenomenon also common in other high-yielding ALA strains. In this study, it is speculated that the enhancement of the ALA production module may have caused more metabolic flux (including carbon flux and energy) to be diverted from cell growth to ALA synthesis, resulting in an imbalance between cell growth and ALA production ( Figure 1 ).

[0181] 6. Construction of recombinant Staphylococcus pasteurianus SpALA

[0182] In this study, gltA, acs, and pyk were selected to accelerate the carbon and energy metabolic flows of S. pasteuri PK8 to enhance cell growth ( Figure 1 The inducible plasmid pRMC2 was double-digested with KpnI and EcoRI to obtain a pRMC2 plasmid fragment. The gltA, acs, and pyk genes were amplified from S. pasteuri PK8 DNA using primers RgltA-F / R, Racs-F / R, and Rpyk-F / R, respectively, to obtain the verified correct gene fragments gltA gene (GeneID: OQ828637, 14 April 2023), acs gene (GeneID: OQ828638, 14 April 2023), and pyk gene (GeneID: OQ828641, 14 April 2023). The pRMC2 plasmid fragment, gltA, acs, and pyk gene fragments were simultaneously connected using seamless cloning, and then transformed into E. coli DH5a competent cells to obtain recombinant E. coli E. coli / pRgap containing the pRgap plasmid. The pRgap plasmid in the recombinant E. coli was extracted and purified for later use.

[0183] A temperature-responsive timing regulation strategy was used to balance ALA production and cell growth in S. pasteuri PK8. In this study, the original replicon in the plasmid pRgap of the S. pasteuri PK8 cell growth module was modified into a temperature-sensitive replicon, which was converted into a temperature-sensitive growth module plasmid pRgap. + gap. Finally, pR + The gap plasmid was integrated into the BRA72S strain to obtain the SpALA strain, which is a strain in which the hemB gene of S. pasteuri PK8 is downregulated and the ALA production module (hemA, pgr7, heml2 and ALAS) and the cell growth module (gltA, acs and pyk) are co-expressed, thus realizing the combination of cell growth and ALA production modules.

[0184] The specific construction steps of the recombinant strain SpALA are as follows: the temperature-sensitive replicon repF (995 bp) in the pnCasSA-BEC plasmid (the nucleotide sequence is sequence 3 in the sequence list) is amplified by PCR using the repF-F / repF-R primers, and the Erm resistance gene (1467 bp) is amplified by PCR using the Erm-F / Erm-R primers (the nucleotide sequence is sequence 4 in the sequence list), and these two gene fragments are used to replace the original replicon and Cm resistance gene of the pRgap plasmid, respectively, to obtain the temperature-sensitive plasmid pR + gap. The recombinant plasmid pR + gap was introduced into strain BRA72S, and the recombinant strain SpALA was finally obtained.

[0185] The SpALA strain obtained above was fermented in two stages. The first 12 hours were fermented at 30°C to ensure the priority growth of cells, and the last 12 hours were fermented at 37°C to focus on ALA production. The shake flask fermentation results showed that ( Figure 2 ), the ALA production of the SpALA strain was as high as 2012.67 mg / L, and the cell growth was almost restored to the level of the wild-type S. pasteuri PK8.

[0186] The SpALA strain was further expanded in a 5L fermenter, and a two-stage fermentation strategy was used for cell growth and ALA production. Figure 4 As shown, the ALA production can reach 3.41g / L (3412.84mg / L), and the bacterial biomass can reach 37 OD 600 , the ALA yield was 26.99 times that of the starting strain S. pasteuri PK8. Compared with the highest ALA yield (0.053 g / L·h) currently produced using a non-model strain (Propionibacterium acidipropionici) (Jiang et al., 2022; Sonhom et al., 2012), the SpALA strain obtained in this study achieved the highest ALA yield produced by a new non-model strain, reaching 0.14 g / L·h. Therefore, temperature-responsive temporal regulation is an effective strategy for synergistically balancing the expression of multiple metabolic modules to increase production. This strategy can also be applied to other PGPRs or to produce more high-value-added compounds using S. pasteuri PK8 as a base strain.

[0187] Example 2: Study on the Growth-Promoting Effect of Recombinant Staphylococcus pasteurianus SpALA

[0188] The SpALA strain not only produced a high yield of ALA, but also significantly (p < 0.01) increased the secretion of siderophores and IAA by 13% and 6%, respectively, compared with S. pasteuri PK8. Figure 5 In (a), on the one hand, this study can separate and purify the high-value-added product - plant growth regulator ALA from the high-yield SpALA fermentation broth. On the other hand, the classic plant growth-promoting properties of SpALA are also effectively improved.

[0189] ALA was extracted and purified according to the following method: ALA was purified from Staphylococcus pasteurianus SpALA fermentation broth using a strong cation exchanger (Seplite LX010). The resin was pretreated according to the manufacturer's instructions and then placed in a column (2 cm × 40 cm) containing distilled water and filled to 100 cm. 3Prior to the adsorption process, the pH of the culture broth was adjusted to 3.0-3.5 with sulfuric acid. The fermentation broth was circulated twice at a flow rate of 10 mL / min. During the elution process, 0.3 M ammonium hydroxide solution was used to release the ALA bound to the resin. After adsorption and elution, the eluate was freeze-dried to obtain a bio-ALA powder with a purity of 80%.

[0190] 1. Study on the germination-promoting effect of Staphylococcus pasteurianus SpALA

[0191] Select healthy and complete corn seeds Zhengdan 958 (purchased from Hefei Hefeng Seed Technology Co., Ltd.), soak them in 70% (v / v) ethanol and shake them gently continuously. After 3 minutes, discard the ethanol; then soak them in 1% sodium hypochlorite solution and shake them gently continuously for 10 minutes. After discarding the sodium hypochlorite solution, rinse the seeds 7 times with sterile saline. The corn seeds were placed with the embryo facing up and divided into 8 groups according to the experimental requirements, with 30 seeds in each group. 30 mL of the following 8 different treatment solutions were added to the culture dish, and placed in a dark environment at 28°C for germination. The germination rate was calculated after 48 hours. The formula for calculating the germination rate is: Germination rate (%) = (number of corn buds with a bud length of about 1 cm / number of 30 seeds) * 100%. The experiment was repeated 3 times. The specific grouping treatments are as follows:

[0192] 1) Con: Corn seeds were directly soaked in 30 mL of sterile saline;

[0193] 2) 5ALA: Corn seeds were soaked in 30 mL of 5 mg / L pure ALA solution;

[0194] 3) PK8 bacteria: Corn seeds were soaked in Staphylococcus pasteurianus PK8 washed with sterile saline and diluted to 10 8 CFU / mL in 30mL bacterial suspension solution;

[0195] 4) PK8 supernatant: Corn seeds were soaked in the following solution: the supernatant of the centrifuged Staphylococcus pasteurianus PK8 fermentation broth was sterilized by filtration through a 0.22 μm sterile filter, and the resulting supernatant was diluted with sterile saline to 30 mL of a 5 mg / L ALA solution;

[0196] 5) PK8 fermentation broth (containing PK8 fermentation supernatant and strain): Corn seeds were soaked in 30 mL of Staphylococcus pasteurianus PK8 fermentation broth, which was directly diluted with sterile saline to a 5 mg / L ALA solution;

[0197] 6) SpALA bacteria: Corn seeds were soaked in sterile saline and washed with Staphylococcus pasteurianus SpALA and diluted to 10 8 CFU / mL in 30mL solution;

[0198] 7) SpALA supernatant: Corn seeds were soaked in the following solution: the supernatant of the centrifuged Staphylococcus pasteurianus SpALA fermentation broth was sterilized by filtration through a 0.22 μm sterile filter, and the resulting supernatant was diluted with sterile saline to a 5 mg / mL solution of SpALA (30 mL).

[0199] 8) SpALA fermentation broth (containing SpALA fermentation supernatant and strain): Corn seeds were soaked in 30 mL of a solution of Staphylococcus pasteurianus SpALA fermentation broth, which was directly diluted with sterile saline to a concentration of 5 mg / L ALA.

[0200] Corn germination research results show that ( Figure 5 In (b), corn seeds treated with the recombinant bacteria SpALA significantly outperformed those treated with the original bacteria PK8 in all three treatments: bacterial solution, supernatant, and fermentation broth. These improvements were 7%, 11%, and 5%, respectively. Direct fermentation broth treatment had the greatest germination-promoting effect, followed by bacterial culture, and finally supernatant treatment. SpALA fermentation broth treatment significantly boosted germination by 109% compared to the sterile saline control.

[0201] 2. Study on the growth promotion and salt-alkali stress resistance of Staphylococcus pasteurianus SpALA

[0202] 20 mL of 1 / 2 MS agar (0.8%) medium was dispensed into glass test tubes (16*150 mm). The saline-alkali stress treatment was performed by adding NaCl with a final concentration of 200 mM to the 1 / 2 MS agar medium, adjusting the pH to 8.5 with sodium bicarbonate, and sterilizing at 121°C for 15 min.

[0203] The germinated corn buds of the above 8 treatments were transferred to glass test tubes containing 1 / 2MS, and the seeds were gently pressed with sterile tweezers to insert the primary roots into the agar. Among them, a part of the corn seeds treated with SpALA fermentation broth and PK8 fermentation broth were sown in glass test tubes of 1 / 2MS under severe saline-alkali stress (200mM NaCl, pH 8.5). Subsequently, a sterile empty glass test tube of the same size was placed mouth to mouth with the test tube containing corn seeds and sealed with a sealing film. The seedlings were placed in an artificial climate chamber at 25°C (light intensity 550μmol / m 2 / s, 50% humidity, 16 h; dark, 50% humidity, 8 h) for 90 h, and the lengths of the aboveground part and roots of the maize seedlings, as well as the fresh weight and dry weight of the whole plant were measured.

[0204] The specific groups are as follows:

[0205] 1) Con: Germinated corn seeds in sterile saline and sown in glass test tubes containing 1 / 2 MS under normal conditions;

[0206] 2) 5ALA: Germinated corn seeds in 5 mg / L ALA sterile saline solution were sown in glass test tubes containing 1 / 2 MS under normal conditions;

[0207] 3)PK8 bacteria: 10 8 CFU / mL PK8 bacteria + germinated corn seeds in sterile saline solution, sown in glass test tubes containing 1 / 2MS under normal conditions;

[0208] 4) PK8 supernatant: Germinate corn seeds in 5 mg / L ALA+PK8 fermentation supernatant and sow in glass test tubes containing 1 / 2 MS under normal conditions;

[0209] 5) PK8 fermentation broth: Germinated corn seeds in 5 mg / L ALA+PK8 fermentation broth were sown in glass test tubes containing 1 / 2 MS under normal conditions;

[0210] 6)SpALA bacteria: 10 8 CFU / mL SpALA bacteria + germinated corn seeds in sterile saline solution, sown in glass test tubes containing 1 / 2MS under normal conditions;

[0211] 7) SpALA supernatant: Germinate corn seeds in the supernatant of 5 mg / L ALA+SpALA fermentation broth and sow them in a glass test tube containing 1 / 2 MS under normal conditions;

[0212] 8) SpALA fermentation broth: Germinated corn seeds in 5 mg / L ALA+SpALA fermentation broth were sown in glass test tubes containing 1 / 2 MS under normal conditions;

[0213] 9) Na8Con: Germinated corn seeds in sterile saline and sown in glass test tubes containing 1 / 2 MS under severe saline-alkali stress;

[0214] 10) Na8PK8 fermentation broth: Germinate corn seeds in 5 mg / L ALA+PK8 fermentation broth (containing PK8 fermentation supernatant and bacterial liquid) and sow in glass test tubes containing 1 / 2 MS under severe saline-alkali stress;

[0215] 11) SpALA fermentation broth: Corn seeds were germinated in 5 mg / L ALA+SpALA fermentation broth (containing SpALA fermentation supernatant and bacterial liquid) and sown in glass test tubes containing 1 / 2 MS under severe saline-alkali stress.

[0216] The results of the resistance of Staphylococcus pasteurianus SpALA to salt-alkali stress are as follows Figure 6As shown, for the growth of corn seedlings, among the 11 treatments, the fermentation liquid is still the best, followed by the bacterial liquid, and finally the supernatant; even the worst SpALA supernatant treatment (SpALA supernatant group) is significantly better than the 5 mg / L ALA aqueous solution (5ALA group) in terms of plant height, root length and whole plant fresh weight of corn seedlings; this shows that the simple ALA treatment only increases the root length, while the SpALA supernatant treatment with the same ALA concentration not only increases the root length, but also significantly promotes plant height. This result also verifies that in addition to secreting more ALA, SpALA also secretes more siderophores and IAA growth-promoting substances. On the other hand, among the 11 treatments, the S. pasteuri SpALA treatment is significantly better than the corresponding S. pasteuri PK8 treatment in terms of corn plant height and root length. Under the three treatments of bacteria, supernatant, and fermentation liquid, the growth-promoting effect of SpALA on corn seedling height is significantly increased by 57%, 26%, and 66% (compared to PK8) respectively. Figure 6 b), the root length was significantly increased by 46%, 23%, and 40% compared with PK8 ( Figure 6 (c) This indicates that the increase in ALA yield and the auxiliary increase in other growth-promoting substances significantly promoted the growth-promoting effect of the PGPR strain.

[0217] Under severe saline-alkali stress, the growth of corn seedlings treated with SpALA fermentation liquid from S. pasteuri was almost completely restored, or even better than that under normal conditions: the root length of corn seedlings treated with SpALA fermentation liquid was significantly (p<0.05) higher than that under normal growth conditions by 33%, and the number of lateral roots was significantly increased. The ALA produced by SpALA is also an acidic substance (pK a =4.05), and its by-product organic acids may also participate in the stress resistance process by directly neutralizing alkaline stress. Under saline-alkali stress, plants can maintain pH stability and iron balance by synthesizing and secreting organic acids.

[0218] The above results show that the PGPR bacteria S. pasteuri SpALA fermentation broth developed in the present invention has the advantages of direct, simple, and efficient application, eliminating complex processing and purification steps. This not only directly utilizes the three growth-promoting factors (ALA, siderophores, and IAA) secreted by the PGPR bacteria SpALA fermentation, which simultaneously enhance the comprehensive growth-promoting and stress-resistance effects, but also directly uses the SpALA fermentation broth to show better growth-promoting and salt-alkali tolerance compared to wild-type S. pasteuri PK8. It also ensures that the presence of PGPR bacteria SpALA exerts a sustainable growth-promoting and stress-resistance effect. The application method is simpler, more effective, and less costly, proposing a new strategy for the direct application of PGPR and its fermentation products in sustainable agriculture.

[0219] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Recombinant Staphylococcus pasteurianus, Features: The recombinant Staphylococcus pasteurianus knocks down the ALA dehydratase gene hemB; the recombinant Staphylococcus pasteurianus overexpresses the glutamyl tRNA reductase gene hemA, the glutamyl-1-semialdehyde aminotransferase gene heml2, the proton gradient regulation 7 gene pgr7 and the 5-aminolevulinic acid synthase gene ALAS, and also contains the citrate synthase gene gltA, the acetyl-CoA synthetase gene acs, and the pyruvate kinase gene pyk.

2. The recombinant Staphylococcus pasteurianus according to claim 1, Features: The hemA gene encodes any of the following proteins: A1) The amino acid sequence encoded by the hemA gene is the protein hemA with the Genbank number WIF29717 (14 April 2023); A2) a protein having hemA activity and having at least 80% identity with the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of A1); A3) connecting a tag to the N-terminus and / or C-terminus of A1) or A2) to obtain a fusion protein having hemA activity; The heml2 gene encodes any of the following proteins: B1) The amino acid sequence encoded by the heml2 gene is the protein heml2 with the Genbank number WIF29719 (14 April 2023); B2) a protein having more than 80% identity with the protein shown in B1) and having heml2 activity obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in B1); B3) connecting a tag to the N-terminus and / or C-terminus of B1) or B2) to obtain a fusion protein having heml2 activity; The pgr7 gene encodes any of the following proteins: C1) The amino acid sequence encoded by the pgr7 gene is the protein pgr7 shown in the Appendix; C2) a protein having pgr7 activity and having more than 80% identity with the protein shown in C1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in C1); C3) connecting a tag to the N-terminus and / or C-terminus of C1) or C2) to obtain a fusion protein having pgr7 activity; The ALAS gene encodes any of the following proteins: D1) The amino acid sequence of the ALAS gene is from the protein ALAS of Rhodobacter sphaeroides 2.4.1GeneID: 67448527, EC number: 2.3.1.37 in NCBI; D2) a protein having ALAS activity and having at least 80% identity with the protein shown in D1) by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in D1); D3) connecting a tag to the N-terminus and / or C-terminus of D1) or D2) to obtain a fusion protein having ALAS activity; The gltA gene encodes any of the following proteins: E1) The amino acid sequence encoded by the gltA gene is the protein gltA with the Genbank number WIF29721 (14 April 2023); E2) a protein having 80% or more identity with the protein shown in E1) and having gltA activity, obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in E1); E3) connecting a tag to the N-terminus and / or C-terminus of E1) or E2) to obtain a fusion protein having gltA activity; The acs gene encodes any of the following proteins: F1) The amino acid sequence encoded by the acs gene is the protein acs with the Genbank number WIF29722 (14 April 2023); F2) a protein having ACS activity and having more than 80% identity with the protein shown in F1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in F1); F3) A fusion protein having ACS activity obtained by connecting a tag to the N-terminus and / or C-terminus of F1) or F2); The pyk gene encodes any of the following proteins: G1) The amino acid sequence encoded by the pyk gene is the protein pyk with the Genbank number WIF29725 (14 April 2023); G2) a protein having pyk activity and having more than 80% identity with the protein shown in G1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in G1); G3) A fusion protein having pyk activity obtained by connecting a tag to the N-terminus and / or C-terminus of G1) or G2); The hemB gene encodes any of the following proteins: H1) The amino acid sequence encoded by the hemB gene is the protein hemB with the Genbank number WIF29720 (14 April 2023); H2) a protein having hemB activity and having at least 80% identity with the protein shown in H1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in H1); H3) A tag is connected to the N-terminus and / or C-terminus of H1) or H2) to obtain a fusion protein with hemB activity.

3. The recombinant Staphylococcus pasteurianus according to claim 1 or 2, Features: The recombinant Staphylococcus pasteurianus is constructed according to the method of claim 4.

4. Method for constructing recombinant Staphylococcus pasteurianus, Features: The method comprises knocking down the hemB gene described in claim 1 or 2 in a recipient Staphylococcus pasteurianus, introducing hemA, heML2, pgr7 and ALAS described in claim 1 or 2 into the recipient Staphylococcus pasteurianus, and introducing gltA, acs, and pyk described in claim 1 or 2 into the recipient Staphylococcus pasteurianus, thereby obtaining a recombinant Staphylococcus pasteurianus with a higher 5-aminolevulinic acid production than that of the recipient bacteria.

5. A method for producing 5-aminolevulinic acid, comprising culturing the recombinant Staphylococcus pasteurianus according to claim 1 or 2 to obtain a fermentation product, and obtaining 5-aminolevulinic acid from the fermentation product.

6. The method of claim 5 or the recombinant Staphylococcus pasteurianus of claim 1 or 2 is used in any of the following: P1. Application in the production of 5-aminolevulinic acid; P2. Application in increasing the production of 5-aminolevulinic acid.

7. The use of the method of claim 5 or the recombinant Staphylococcus pasteurianus described in claim 1 or 2 in any of the following: (a1) promoting plant growth; (a2) preparing products for promoting plant growth; (a3) Producing IAA; (a4) preparing an IAA-producing product; (a5) siderophore production; (a6) preparing a siderophore-producing product; (a7) promoting plant germination; (a8) Improve the ability of plants to resist salt-alkali stress.

8. A method for promoting plant growth, comprising the following steps: applying the recombinant Staphylococcus pasteurianus according to claim 1 or 2 to a test plant, thereby promoting plant growth.

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