Decarboxylase mutants in indolepyruvate pathway, mutant combinations thereof and applications in indoleacetic acid production

Abstract

This invention belongs to the field of microbial genetic engineering and metabolic engineering technology, specifically relating to decarboxylase mutants in the indolepyruvate pathway, their mutant combinations, and their application in the production of indoleacetic acid. A decarboxylase mutant was obtained by mutating glycine at position 255 of the indolepyruvate decarboxylase kivD derived from *Lactococcus lactis* to tyrosine, and serine at position 386 to glutamic acid. kivD G255Y / S386E The applicant expressed kivD freely in the engineered recombinant Bacillus licheniformis strain BLI-2. G255Y / S386E The strain BlI-3 was obtained, and the indoleacetic acid (IAA) yield of BlI-3 reached 675.6 mg / L, which was 70.09% higher than that of BlI-2 and 258.44% higher than that of BlI-1, significantly improving the level of IAA synthesis by the host strain.

Description

Technical Field

[0001] This invention relates to the fields of microbial genetic engineering and metabolic engineering, specifically to decarboxylase mutants in the indolepyruvate pathway, combinations of mutants thereof, and their application in the production of indoleacetic acid. Background Technology

[0002] Indole-3-acetic acid (IAA) is a key plant hormone that regulates cell division, development, and metabolism. At the cellular level, IAA drives plant cell development by synergistically regulating cell wall dynamics and cell cycle progression. Plant organ and individual development are significantly affected by IAA concentration, with different organs exhibiting marked differences in their response thresholds. Roots are particularly sensitive to fluctuations in IAA concentration. Studies have shown that both excessively high and low IAA concentrations inhibit root elongation. Indole-3-acetic acid (IAA) also plays a crucial role in plant-microbe interactions. Many plant rhizosphere microorganisms can synthesize IAA, promoting plant growth and enhancing plant tolerance to abiotic stresses such as drought and salinity by regulating IAA levels. Besides its role as a plant growth regulator, IAA can also be used as a component of pesticides and herbicides. Indole-3-acetic acid exhibits high selectivity and efficiency, effectively suppressing or killing some harmful weeds and pests, demonstrating good efficacy in controlling weeds and pests in farmland. Therefore, large-scale production of IAA is of great significance to the development of modern agriculture.

[0003] Many microorganisms can synthesize IAA, with rhizosphere bacteria that interact with plants playing a dominant role. Numerous studies have confirmed that bacterial IAA synthesis pathways include tryptophan-dependent and tryptophan-independent pathways, with the indolepyruvate pathway (IPA pathway) being the main pathway for tryptophan-dependent IAA biosynthesis widely found in bacteria. The IPA pathway mainly achieves the conversion of tryptophan to IAA through three enzymatic reactions: (1) L-tryptophan is catalyzed by aminotransferase to generate indole-3-pyruvate (IPyA); (2) indolepyruvate decarboxylase catalyzes the decarboxylation of IPyA to form indole-3-acetaldehyde (IAAld); (3) IAA is oxidized by aldehyde dehydrogenase to generate IAA.

[0004] Wild-type plant rhizosphere growth-promoting bacteria generally have low levels of IAA synthesis, making large-scale application in production impossible. Summary of the Invention

[0005] The purpose of this invention is to provide a decarboxylase mutant kivD in the indolepyruvate pathway. G255Y / S386E The decarboxylase mutant kivD G255Y / S386E The amino acid sequence of the protein is shown in SEQ ID NO.8, and the polynucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO.7.

[0006] Another object of the present invention is to provide a mutant combination comprising the tryptophan transaminase mutant AspC. A110Q / N30A and decarboxylase mutant kivD G255Y / S386E The tryptophan transaminase mutant AspC A110Q / N30A The amino acid sequence of the protein is shown in SEQ ID NO.4, and the decarboxylase mutant kivD G255Y / S386E The amino acid sequence of the protein is shown in SEQ ID NO.8.

[0007] The final objective of this invention is to provide a decarboxylase mutant kivD G255Y / S386E Or the application of the above mutant combinations in the production of indoleacetic acid (IAA).

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] To address the problem of low indoleacetic acid yield in existing technologies, this invention provides a decarboxylase mutant, kivD. G255Y / S386E And its relationship with the tryptophan transaminase mutant AspC A110Q / N30A The combination of these components is used in the biofermentation production of indoleacetic acid. The applicant mutated alanine at position 110 of the tryptophan transaminase aspC (SEQ ID NO.2) derived from *E. coli* to glutamine and asparagine at position 30 to alanine, obtaining the tryptophan transaminase mutant AspC of this invention. A110Q / N30A As shown in SEQ ID NO.4, the indolepyruvate decarboxylase kivD from Lactococcus lactis was modified by mutating glycine at position 255 to tyrosine and serine at position 386 to glutamic acid, resulting in the decarboxylase mutant kivD. G255Y / S386E Its amino acid sequence is shown in SEQ ID NO.8.

[0010] The scope of protection of this invention also includes:

[0011] The fusion protein obtained by fusing the mutant protein shown in SEQ ID NO.8 with a protein purification tag.

[0012] The gene encoding the mutant protein or fusion protein shown in SEQ ID NO.8.

[0013] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0014] The use of the mutant protein shown in SEQ ID NO.8, the above-mentioned fusion protein, the above-mentioned encoding gene, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the ex vivo recombinant cell in the preparation of decarboxylase mutants.

[0015] The use of the mutant protein shown in SEQ ID NO.8, the above-mentioned fusion protein, the above-mentioned encoding gene, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the ex vivo recombinant cell in the production of indoleacetic acid.

[0016] A method for increasing the activity of decarboxylase includes mutating glycine at position 255 of SEQ ID NO.6 to tyrosine and serine at position 386 to glutamic acid.

[0017] A recombinant protein combination comprising protein A and protein B, wherein protein A is one of the mutant protein shown in SEQ ID NO.4 or a fusion protein obtained by fusing it with a protein purification tag, and protein B is one of the variant protein shown in SEQ ID NO.8 or a fusion protein obtained by fusing it with a protein purification tag.

[0018] The gene encoding the above recombinant protein combination.

[0019] Expression cassettes, recombinant vectors, recombinant microorganisms, or ex vivo recombinant cells containing gene encoding recombinant protein combinations.

[0020] A method for preparing indoleacetic acid using the above-mentioned mutant protein, fusion protein, or recombinant protein combination includes introducing or integrating an expression vector expressing the mutant protein shown in SEQ ID NO. 8 or its fusion protein into the genetically engineered Bacillus licheniformis strain B1I-2, wherein the genetically engineered Bacillus licheniformis strain B1I-2 expresses tryptophan transaminase aspC (amino acid sequence shown in SEQ ID NO. 2) or the tryptophan transaminase mutant AspC. A110Q / N30A Recombinant Bacillus licheniformis containing indolepyruvate decarboxylase kivD (amino acid sequence shown in SEQ ID NO.4), indolepyruvate decarboxylase kivD (amino acid sequence shown in SEQ ID NO.6), and indoleacetaldehyde dehydrogenase dhaS (amino acid sequence shown in SEQ ID NO.10).

[0021] In the preferred embodiment of the above-described method, the recombinant Bacillus licheniformis is Bacillus licheniformis DW2 (CN116656712A).

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention focuses on the modification and screening of three synthases in the indolepyruvate pathway, obtaining mutants with significantly increased substrate conversion efficiency and IAA synthesis, AspC. A110Q / N30A kivD G255Y / S386E dhaS P169I / G271W The mutant AspC A110Q / N30A By integrating recombinant Bacillus licheniformis engineered strain BLI-1, which expresses wild-type tryptophan transaminase aspC, indolepyruvate decarboxylase kivD, and indoleacetaldehyde dehydrogenase dhaS, strain BLI-2 was obtained. The IAA yield of this strain was increased by 110.73% compared with BLI-1, reaching 397.2 mg / L.

[0024] KivD was expressed free in BLI-2. G255Y / S386E dhaS P169I / G271W Strains BlI-3 and BlI-4 were obtained. After adding 1 g / L tryptophan to the fermentation medium, the indoleacetic acid yields of BlI-3 and BlI-4 reached 675.6 mg / L and 585.7 mg / L, respectively, representing increases of 70.09% and 47.46% compared to BlI-2, and increases of 258.44% and 210.73% compared to BlI-1.

[0025] This application significantly improves the level of IAA synthesis by the host strain by utilizing the superposition of two effects: increased catalytic activity of mutant enzymes and increased copy number of enzymes (dosage effect). Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art, and the reagents or materials, unless otherwise specified, are all derived from commercial sources.

[0027] The culture medium, IAA fermentation conditions, and substance detection methods involved in the embodiments of this invention are as follows:

[0028] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0029] LB solid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar powder 15 g / L.

[0030] LANDY medium: glucose 20 g / L, yeast extract 5 g / L, L-phenylalanine 0.002 g / L, L-glutamate sodium 5 g / L, potassium chloride 0.5 g / L, potassium dihydrogen phosphate 1 g / L, manganese sulfate tetrahydrate 0.005 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.00015 g / L, copper sulfate heptahydrate 0.00016 g / L, pH 7.0-7.2.

[0031] Fermentation conditions for IAA production by Bacillus licheniformis: The seed culture was inoculated at a rate of 1% into a conical flask containing 50 mL of Landy medium, and cultured in a shaker at 120 r / min and 30℃ in the dark for 72 h.

[0032] IAA detection method: Spectrophotometry was used for detection. The sample preparation steps are as follows: Centrifuge 2 mL of fermentation broth at 12000 rpm for 10 min, then take 1 mL of supernatant (dilute as needed) and add 1 mL of colorimetric reagent (H2SO4 772.4 g / L, FeCl3 12 g / L, freshly prepared and used). After reacting in the dark for 30 min, the OD value is measured using a spectrophotometer. 530 The yield of indole-3-acetic acid in the fermentation sample was calculated based on the standard curve.

[0033] IAA synthesis precursor and intermediate detection method: 2 mL of fermentation broth was centrifuged at high speed (12000 rpm for 10 min), and the supernatant was collected. The supernatant was filtered through a 0.22 μm pore size filter membrane, and the contents of tryptophan and indolepyruvate were detected by HPLC.

[0034] Example 1:

[0035] Construction of Bacillus licheniformis engineered strain BLI-1 for IAA production:

[0036] The applicant integrated three key enzymes of the IPA pathway into Bacillus licheniformis DW2: the tryptophan transaminase gene aspC (SEQ ID NO.1, encoded protein as SEQ ID NO.2) from Escherichia coli, the indolepyruvate decarboxylase gene kivD (SEQ ID NO.5, encoded protein as SEQ ID NO.6) from Lactococcus lactis, and the indoleacetaldehyde dehydrogenase gene dhaS (SEQ ID NO.9, encoded protein as SEQ ID NO.10) from Bacillus licheniformis.

[0037] The gene integration expression of the engineered Bacillus licheniformis strain BLI-1 was achieved based on homologous recombination, and the construction methods of the integration plasmids for the three genes aspC, kivD, and dhaS were similar.

[0038] The construction of the integrated gene kivD plasmid is illustrated using this example. Using *Bacillus licheniformis* DW2 as a template, primers yvnA-F1 / R1 and yvnA-F2 / R2 were used to amplify the upstream and downstream homologous regions of the gene yvnA. Using the pHY300PLK plasmid carrying the R55 promoter as a template, primer R55-F / R was used to amplify the enhanced R55 promoter. Using *Lactococcus lactis* genomic DNA as a template, primer yvnA-kivD-F / R was used to amplify the gene kivD. The R55 promoter was ligated to its upstream homologous arm, and the downstream homologous arm to the gene kivD, using SOE-PCR. The two SOE fragments and the T2 backbone were then digested and ligated using recombinases, and transformed into *E. coli* DH5α. The transformants were verified by colony PCR and nucleic acid sequencing. Successful verification yielded the integrated expression plasmid T2-yvnA::kivD.

[0039] The integration expression plasmids T2-oxdC::dhaS and T2-yugK::aspC were constructed in the same manner.

[0040] Taking the integration of kivD into the genome of Bacillus licheniformis DW2 as an example: the recombinant plasmid T2-yvnA::kivD was introduced into DW2 competent cells by electroporation. After resuscitation culture, kanamycin-resistant transformants were screened. Candidate strains were initially screened by PCR using primers T2-F / R. Positive clones were confirmed by sequencing and entered the homologous recombination stage. First, single-exchange strains were screened. The verified transformants were continuously passaged for three generations. Then, colony PCR was performed for secondary verification using primers T2-F / yvnA-kivD-YR or yvnA-kivD-YF / T2-R. After obtaining stable single-exchange strains, double exchange was induced in antibiotic-free medium. Finally, the final product was specifically amplified using the verification primer yvnA-kivD-YF / YR. After electrophoresis and sequencing confirmed that the kivD gene was accurately integrated into the yvnA site, the target engineered bacterium DW2-yvnA::kivD was successfully obtained.

[0041] Furthermore, aspC and dhaS were integrated by combining expression plasmids T2-oxdC::dhaS and T2-yugK::aspC, respectively, and the integration method was similar to the method described above.

[0042] Ultimately, a Bacillus licheniformis strain integrating the three genes was obtained, which was named the genetically engineered strain BLI-1.

[0043] The genetically engineered strain BLI-1 was fermented on LANDY medium, and the IAA yield was 188.5 mg / L. The accumulation of tryptophan and indolepyruvate was 950.4 mg / L and 105.3 mg / L, respectively. Tryptophan was not effectively converted and the IAA yield was low.

[0044] The primer sequences used for gene integration are as follows:

[0045] yvnA-F1: GATCTTTTCTACGAGCTCATTCGACTGGTGCATTGC, as shown in SEQ ID NO.13.

[0046] yvnA-R1: GCAAACAGCCCCCCCCACCAAAAGGCATATGATGCA, as shown in SEQ ID NO.14;

[0047] yvnA-F2: GCTGGACCGTCATCATTATCAAGAGGAGCAGCGAAT, as shown in SEQ ID NO.15.

[0048] yvnA-R2: AACGAATTCCTGCAGCCCAAGACTTCGTTTACCGTG, as shown in SEQ ID NO.16;

[0049] R55-F: GTGGGGGGGGCTGTTTGC, as shown in SEQ ID NO.17.

[0050] R55-R: ACAAATCTCCCCCTTTGT, as shown in SEQ ID NO.18;

[0051] yvnA-kivD-F:ACAAAGGGGGAGATTTGTATGTATACAGTAGGAGAT,as shown in SEQ ID NO.19

[0052] yvnA-kivD-R:TCCGTCCTCTCTGCTCTTTTATGATTTATTTTGTTC,as shown in SEQ ID NO.20;

[0053] yvnA-kivD-YF:TAGCGGACAGGAGGAGGCA, as shown in SEQ ID NO.21,

[0054] yvnA-kivD-YR:TCCACAACTGTGTAGGAGCGG, as shown in SEQ ID NO.22;

[0055] oxdC-dhaS-F:AGAAAGGAGGAATATATATTGACAAACATGAGTTCA, as shown in SEQ ID NO.23.

[0056] oxdC-dhaS-R:TCCGTCCTCTCTGCTCTTTTATTCGCCTGTATGAAT, as shown in SEQ ID NO.24;

[0057] oxdC-dhaS-YF: ATCGTCCCC, as shown in SEQ ID NO.25.

[0058] oxdC-dhaS-YR: CAGGTAGACGCCGTAAGTC, as shown in SEQ ID NO.26;

[0059] yugK-aspC-F:TATATATTCCTCCTTTCTAATATAC,as shown in SEQ ID NO.27,

[0060] yugK-aspC-R: AAGAGCAGAGAGGACGGATTTCC, as shown in SEQ ID NO. 28;

[0061] yugK-aspC-YF: CTGTCGGTTTCATTCACG, as shown in SEQ ID NO.29

[0062] yugK-aspC-YR: CGGGATCAGCAAAAAAAG, as shown in SEQ ID NO.30.

[0063] Example 2:

[0064] Tryptophan transaminase mutant AspC A110Q / N30A Acquisition and construction:

[0065] Based on the structure of wild-type tryptophan transaminase (SEQ ID NO.2), the applicant designed hundreds of single-point mutants, selected dozens of these single-point mutants (a partial list of mutant sites is shown in Table 1), integrated them into the pHY300PLK vector, and transformed it into BLI-1 for free expression experiments, to carry the empty pHY300PLK plasmid and pHY-P, respectively. RBS6 Using BLI-1 of -aspC as a control, and IAA yield measured during fermentation as a metric, the AspC single mutant with increased IAA yield compared to the control was finally screened. A110Q and AspC N30A The applicant fused these two single mutations into a single mutant, preparing AspC. A110Q / N30AThe mixture was then recombined into the pHY300PLK vector and transformed into BLI-1. Fermentation experiments showed that the double mutant had a better effect on increasing IAA yield compared with the tryptophan transaminase single mutant.

[0066] Table 1. Partial Single-Point Mutant Sites of Tryptophan Transaminase AspC

[0067]

[0068] The amino acid mutation positions in the table above are based on the sequence shown in SEQ ID NO.2.

[0069] Using *E. coli* as a template, the aspC gene was amplified using aspC-F / R; using *Bacillus subtilis* 168 as a template, the RBS6 promoter was amplified using primers RBS6-F / R; using *Bacillus licheniformis* WX-02 (CN121896189A) as a template, the TamyL terminator of the amylase amyL gene was amplified using primers TamyL-F / R; using the pHY300PLK empty plasmid as a template, the vector backbone was amplified using primers pHY-GF / GR. The promoter RBS6, the aspC gene, and the terminator TamyL were ligated using SOE-PCR. The products were recovered and purified, then digested and ligated with recombinases, and transformed into competent *E. coli* cells. Finally, positive transformants were verified and sequenced; those with correct results were used to obtain the free expression vector pHY-P. RBS6 -aspC.

[0070] Design mutation site-specific primers (aspC) A110Q -F / R), with pHY-P RBS6 Using the -aspC plasmid as a template, primers (RBS6-F / aspC) were used. A110Q -R and aspC A110Q The DNA fragment was amplified using a combination of primers pHY-F / TamyL-R, and the vector backbone was amplified using primers pHY-GF / GR. Finally, the two recovered and purified fragments were ligated to the backbone and calcium-transferred into E. coli DH5α. Transformants with correct bands were validated using the validation primers (pHY-F / R) and subjected to plasmid sequencing. If the sequencing results were correct, the transaminase single mutant expression vector pHY-P... RBS6 -aspC A110Q Successfully built.

[0071] Design mutation site-specific primers (aspC) N30A -F / R), with pHY-P RBS6 -aspC A110Q Using plasmid as a template, primers (RBS6-F / aspC) were used. N30A -R and aspCN30A The DNA fragment was amplified using a combination of primers pHY-F / TamyL-R, and the vector backbone was amplified using primers pHY-GF / GR. Finally, the two recovered and purified fragments were ligated to the backbone and calcium-transferred into E. coli DH5α. After verification that the primers (pHY-F / R) were correct, the tryptophan transaminase mutant AspC was successfully constructed. A110Q / N30A expression vector pHY-P RBS6 -aspC A110Q / N30A The expression vector expresses the tryptophan transaminase mutant AspC. A110Q / N30A It includes the amino acid sequence shown in SEQ ID NO.4, and the polynucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO.3.

[0072] plasmid pHY-P RBS6 -aspC A110Q / N30A Genetic transformation was performed into the engineered Bacillus licheniformis strain BLI-1, and fermentation was carried out in LANDY medium. The residual tryptophan was measured to be 576.9 mg / L, the tryptophan utilization rate was 39.3%, the indolepyruvate accumulation was 206.8 mg / L, and the IAA yield was 284.9 mg / L.

[0073] The primer sequences required for constructing the double mutant are as follows:

[0074] pHY-F: CTTTTTTCAGGAATCATTGTCATTAGTTGGC, as shown in SEQ ID NO.31.

[0075] pHY-R: GCCAGGGGGAAACGCCTGGTATCTTTATAG, as shown in SEQ ID NO.32;

[0076] RBS6-F: TTTGGGTGTGGTATAATT, as shown in SEQ ID NO.33.

[0077] RBS6-R: TTCTAGCTTCTTCCTGAC, as shown in SEQ ID NO.34;

[0078] TamyL-F: AAAGAGCAGAGAGGACGGA, as shown in SEQ ID NO.35,

[0079] TamyL-R: CGCAATAATGCCGTCGCAC, as shown in SEQ ID NO.36;

[0080] pHY-GF: AAAAAGGATCAATTTTGAACTCTCTC, as shown in SEQ ID NO.37.

[0081] pHY-GR: TGATCCTTCCTCCTTTAGATCTGC, as shown in SEQ ID NO.38;

[0082] aspC-F: CAATAAGGGGAAGGATCAATGTTTGAGAACCATTAC, as shown in SEQ ID NO.39.

[0083] aspC-R: CCTCTCGTCGCTCCTTTTTTACAGCATACTCACGCA, as shown in SEQ ID NO.40;

[0084] aspC A110Q -F: CTCGCACGCAACAGACTCCGGGGGGCACTGGCGCAC, as shown in SEQ ID NO.41.

[0085] aspC A110Q -R: GAGTCTGTTGCGTGCGAGCACGTTTGTCATTGATCA, as shown in SEQ ID NO.42;

[0086] aspC N30A -F: AGTTCCTACGAAAAAAACTTTGGCCTGTACAACGAG, as shown in SEQ ID NO.43.

[0087] aspC N30A -R: GTTTTTTTCGTAGGAACTGGCAACAATCAGCTCTTT, as shown in SEQ ID NO.44.

[0088] Example 3:

[0089] Integrates aspC A110Q / N30A Construction of the Bacillus licheniformis engineered strain BLI-2

[0090] Using Bacillus licheniformis DW2 as a template, the upstream and downstream homologous regions of the gene xpt were amplified using primers xpt-F1 / R1 and xpt-F2 / R2, respectively; pHY-P RBS6 -aspC A110Q / N30A As a template, use primers xpt-aspC A110Q / N30A -F / R amplification gene aspC A110Q / N30A SOE-PCR was used to correlate the upstream and downstream homologous arms with the aspC gene. A110Q / N30AThe two SOE fragments and the T2 backbone were ligated, then digested and ligated with recombinases, and finally transformed into E. coli DH5α. The transformants were verified by colony PCR and nucleic acid sequencing. Successful verification yielded the integration expression plasmid T2-xpt::aspC. A110Q / N30A .

[0091] Plasmid T2-xpt:: aspC A110Q / N30A The kanamycin-resistant transformant was introduced into Bacillus licheniformis BLI-1 competent cells via electroporation. After resuscitation and culture, kanamycin-resistant transformants were screened. Candidate strains were initially screened by PCR using primers T2-F / R. Positive clones were confirmed by sequencing and then entered the homologous recombination stage. Finally, the primer xpt-aspC was validated. A110Q / N30A -YF / YR was used for final product-specific amplification, and aspC was confirmed by electrophoresis and sequencing. A110Q / N30A After the gene was precisely integrated into the xpt site, the aspC gene was successfully integrated. A110Q / N30A The genetically engineered bacterium BLI-2.

[0092] The genetically engineered strain BLI-2 was fermented on LANDY medium. The residual tryptophan in the fermentation broth was 180.10 mg / L, with a utilization rate of 81.05%. The accumulation of indolepyruvate was 340.6 mg / L, and the yield of IAA was 397.2 mg / L.

[0093] The primer sequences used are as follows:

[0094] xpt -F1: TGAACCGATCAGAAAGTGGC, as shown in SEQ ID NO.45.

[0095] xpt -R1: TAAACCCCTCCGTTCATCAA, as shown in SEQ ID NO.46;

[0096] xpt -F2:CATTATTGCCGATGGAAAAGTTAC, as shown in SEQ ID NO.47.

[0097] xpt -R2: CCAATCCTGTTGTAAAACGA, as shown in SEQ ID NO.48;

[0098] xpt-aspC A110Q / N30A -F: AGAAAGGAGGAATATATAATGTTTGAGAACATTACC, as shown in SEQ IDNO.49,

[0099] xpt-aspC A110Q / N30A-R: TCCGTCCTCTCTGCTCTTTTACAGCACTGCCACAAT, as shown in SEQ ID NO.50;

[0100] xpt-aspC A110Q / N30A -YF: GCGGCGATTTCGGTTATT, as shown in SEQ ID NO.51.

[0101] xpt-aspC A110Q / N30A -YR: ACTTTTCCTATCAGAGCG, as shown in SEQ ID NO.52;

[0102] T2-F: ATGTGATAACTCGGCGTA, as shown in SEQ ID NO.53

[0103] T2-R: GCAGAGCAGCAGATTACGC, as shown in SEQ ID NO.54.

[0104] Example 4:

[0105] indolepyruvate decarboxylase mutant kivD G255Y / S386E Acquisition and construction:

[0106] With tryptophan transaminase mutant AspC A110Q / N30A Similarly, this mutant also underwent extensive screening.

[0107] expression vector pHY-P RBS6 The construction method of -kivD is the same as in Example 2, except that the aspC gene is replaced with the kivD gene for construction.

[0108] Design mutation site-specific primers (kivD) G255Y -F / R), with pHY-P RBS6 Using the -kivD plasmid as a template, primers (RBS6-F / kivD) were used. G255Y -R and kivD G255Y The DNA fragment was amplified using a combination of primers pHY-F / TamyL-R, and the vector backbone was amplified using primers pHY-GF / GR. Finally, the two purified fragments were ligated to the backbone and calcium-transferred into E. coli DH5α. Amplification and sequencing were performed using the validation primers (pHY-F / R) to confirm the transaminase single mutant expression vector pHY-P. RBS6 - kivD G255Y Successfully built.

[0109] Design mutation site-specific primers (kivD) Ser386Glu - F / R), with pHY-P RBS6 - kivDG255Y Using plasmid as a template, primers (RBS6-F / kivD) were used. Ser386Glu -R and kivD Ser386Glu The DNA fragment was amplified using a combination of primers pHY-F / TamyL-R, and the vector backbone was amplified using primers pHY-GF / GR. Finally, the two recovered and purified fragments were ligated to the backbone and calcium-transferred into E. coli DH5α. After verification that the primers (pHY-F / R) were correct, the indolepyruvate decarboxylase mutant kivD was successfully constructed. G255Y / S386E expression vector pHY-P RBS6 -kivD G255Y / S386E .

[0110] plasmid pHY-P RBS6 -kivD G255Y / S386E Genetic transformation was performed into the engineered Bacillus licheniformis strain BLI-1 and fermented in LANDY medium. The residual tryptophan was measured to be 675.3 mg / L, with a utilization rate of 28.94%. The accumulation of indolepyruvate decreased to 114.1 mg / L, and the amount of IAA synthesized was 335.1 mg / L.

[0111] The primer sequences required for constructing the double mutant are as follows:

[0112] kivD-F: CTAAAGGAGGAAGGATCAATGTATACAGTAGGAGAT, as shown in SEQ ID NO.55.

[0113] kivD-R: CCGTCCTCTCTGCTCTTTTTATGATTTATTTTGTTC, as shown in SEQ ID NO.56;

[0114] kivD G255Y -F: TTCATTTTTATATATCTATAATGGTAAACTCTCAGA, as shown in SEQ ID NO.57.

[0115] kivD G255Y -R: TAGATATATAAAAATGAAGGGAGAGCTTCATCAACT, as shown in SEQ ID NO.58;

[0116] kivD Ser386Glu -F: GCGCTTCAGAAATTTTCTTAAAACCAAAGAGTCATT, as shown in SEQ ID NO.59.

[0117] kivD Ser386Glu-R: AAGAAAATTTCTGAAGCGCCAAAGAATGATGTCCCT, as shown in SEQ ID NO.60.

[0118] Example 5:

[0119] Contains plasmid pHY-P RBS6 -kivD G255Y / S386E Construction of genetically engineered strain BLI-3:

[0120] Electroporation transformation technology was used to convert the recombinant plasmid pHY-P RBS6 -kivD G255Y / S386E Bacillus licheniformis BLI-2 competent cells were introduced, and positive transformants were obtained after resuscitation culture. After colony PCR amplification, agarose gel electrophoresis and nucleic acid sequencing analysis confirmed the correct gene sequence, thus obtaining the genetically engineered strain BLI-3 carrying the target plasmid.

[0121] Since insufficient tryptophan supply severely restricts IAA synthesis in BLI-3, an additional 1 g / L of tryptophan was added to the LANDY fermentation medium. After inoculation with the genetically engineered strain BLI-3, fermentation was carried out. After fermentation, the residual tryptophan in the fermentation broth was measured to be 629.8 mg / L, with a utilization rate of 67.71%, and the accumulation of indolepyruvate was reduced to 83.5 mg / L.

[0122] The results showed that the indolepyruvate decarboxylase mutant kivD G255Y / S386E The expression of this substance facilitates the conversion of indolepyruvate to indoleacetaldehyde and increases IAA production to 675.6 mg / L.

[0123] Example 6:

[0124] Indoleacetaldehyde dehydrogenase mutant dhaS P169I / G271W Construction:

[0125] With tryptophan transaminase mutant AspC A110Q / N30A Similarly, this mutant also underwent extensive screening.

[0126] expression vector pHY-P RBS6 The construction method of -dhaS is the same as in Example 2, except that the aspC gene is replaced with the dhaS gene for construction.

[0127] Design mutation site-specific primers (dhaS) P169I -F / R), with pHY-P RBS6 Using the dhaS plasmid as a template, primers (RBS6-F / dhaS) were used... P169I -R and dhaS P169IThe DNA fragment was amplified using a combination of primers pHY-F / TamyL-R, and the vector backbone was amplified using primers pHY-GF / GR. Finally, the two purified fragments were ligated to the backbone and calcium-transferred into E. coli DH5α. Amplification and sequencing were performed using the validation primers (pHY-F / R) to confirm the transaminase single mutant expression vector pHY-P. RBS6 - dhaS P169I Successfully built.

[0128] Design mutation site-specific primers (dhaS) Gly271Trp - F / R), with pHY-P RBS6 - dhaS P169I Using plasmid as a template, primers (RBS6-F / dhaS) were used. Gly271Trp -R and dhaS Gly271Trp The DNA fragment was amplified using a combination of primers pHY-F / TamyL-R, and the vector backbone was amplified using primers pHY-GF / GR. Finally, the two recovered and purified fragments were ligated to the backbone and calcium-transferred into E. coli DH5α. After verification that the primers (pHY-F / R) were correct, the indolepyruvate decarboxylase mutant dhaS was successfully constructed. P169I / G271W expression vector pHY-P RBS6 -dhaS P169I / G271W .

[0129] plasmid pHY-P RBS6 -dhaS P169I / G271W Genetic transformation was performed into Bacillus licheniformis DW2 and fermented in LANDY medium. The residual tryptophan was 607.2 mg / L, with a utilization rate of 36.11%. The accumulation of indolepyruvate was 138.0 mg / L, and the synthesis of IAA was 387.6 mg / L.

[0130] The primer sequences required for constructing the double mutant are as follows:

[0131] dhaS-F: AAGGAAACTAAGGATGGCATTGACAAAGAGTCATTC, as shown in SEQ ID NO.61,

[0132] dhaS-R: CATCTCGTCTGCTTCTCTTTTCCTTAGCCTGTATGA, as shown in SEQ ID NO.62;

[0133] dhaS P169I -F: CGGGCAAATTATCATCTGGAACTTCCCGCTTCTGAT, as shown in SEQ ID NO.63

[0134] dhaSP169I -R: GTTCCAGATGATAATTTGCCCGACAACTCCGACAGG, as shown in SEQ ID NO.64;

[0135] dhaS Gly271Trp -F: TGAACTCTGGGGAAAGTCGCCGAATATCATCCTTCC, as shown in SEQ ID NO.65

[0136] dhaS Gly271Trp -R: GACTTTCCCCAGAGTTCAAGGGTCACCCGTTTAACC, as shown in SEQ ID NO.66.

[0137] Example 7:

[0138] Contains plasmid pHY-P RBS6 -dhaS P169I / G271W Construction of the genetically engineered strain BLI-4:

[0139] Electroporation transformation technology was used to convert the recombinant plasmid pHY-P RBS6 -dhaS P169I / G271W Bacillus licheniformis BLI-2 competent cells were introduced, and positive transformants were obtained after resuscitation culture. After colony PCR amplification, agarose gel electrophoresis and nucleic acid sequencing analysis confirmed the correct gene sequence, thus obtaining the genetically engineered strain BLI-4 carrying the target plasmid.

[0140] Due to insufficient tryptophan supply severely restricting IAA synthesis in BLI-4, an additional 1 g / L of tryptophan was added to the LANDY fermentation medium. After inoculation with the genetically engineered strain BLI-4, fermentation was carried out. After fermentation, the residual tryptophan in the fermentation broth was measured at 589.4 mg / L, with a utilization rate of 69.78%, and the indolepyruvate accumulation was 132.8 mg / L. The indolepyruvate decarboxylase mutant dhaS... P169I / G271W The expression of this substance increased IAA production to 585.7 mg / L.

[0141] The results of IAA production by fermentation of genetically engineered bacteria BlI-1 to BlI-4 are shown in Table 2.

[0142] Table 2. Tryptophan utilization, IAA yield, and intermediate metabolite content of the strains

[0143] .

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A synthetically produced decarboxylase mutant protein, wherein the amino acid sequence of the decarboxylase mutant protein is shown in SEQ ID NO.

8.

2. The fusion protein obtained by fusing the mutant protein shown in SEQ ID NO.8 with a protein purification tag.

3. The gene encoding the mutant protein or fusion protein shown in SEQ ID NO.

8.

4. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the gene encoding as described in claim 3.

5. The use of the mutant protein of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, an expression cassette having the encoding gene of claim 3, a recombinant vector, a recombinant microorganism, or an ex vivo recombinant cell in the production of indoleacetic acid.

6. A recombinant protein combination comprising protein A and protein B, wherein protein A is one of the mutant protein shown in SEQ ID NO.4 or a fusion protein obtained by fusing it with a protein purification tag, and protein B is one of the variant protein shown in SEQ ID NO.8 or a fusion protein obtained by fusing it with a protein purification tag.

7. The gene encoding the recombinant protein combination of claim 6.

8. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the recombinant protein combination encoding gene of claim 6.

9. A method for preparing indole acetic acid by using the mutant protein of claim 1, the fusion protein of claim 2 or the recombinant protein of claim 6, comprising introducing or integrating an expression vector expressing the mutant protein shown in SEQ ID NO. 8 or the fusion protein of claim 2 into a genetically engineered Bacillus licheniformis BlI-2, wherein the genetically engineered Bacillus licheniformis BlI-2 expresses tryptophan transaminase aspC, tryptophan transaminase mutant AspC A110Q / N30A , indolepyruvate decarboxylase kivD and indoleacetaldehyde dehydrogenase dhaS. The amino acid sequence of the tryptophan transaminase aspC is shown as SEQ ID NO. 2, the amino acid sequence of the tryptophan transaminase mutant AspC A110Q / N30A The amino acid sequence of the indolepyruvate decarboxylase kivD is shown as SEQ ID NO. 6, and the amino acid sequence of the indoleacetaldehyde dehydrogenase dhaS is shown as SEQ ID NO.

10.

10. The application according to claim 9, wherein the recombinant Bacillus licheniformis is Bacillus licheniformis DW2.

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