A recombinant bacterium catalyzing the production of rebaudioside D from stevioside crude sugar and its application

Abstract

The present invention belongs to the technical field of microorganisms and genetic engineering, and specifically relates to a recombinant bacterium that catalyzes stevioside crude sugar to produce rebaudioside D and its application. The recombinant bacterium at least expresses ABC transporter, glycosyltransferase and sucrose synthase; wherein the expression mode of the ABC transporter is constitutive expression, the glycosyltransferases are UGT76G1 and UGTsl2mu, and the sucrose synthase is Atsus1. The recombinant bacterium can directly use stevioside crude sugar as a substrate, and catalyze stevioside STV and rebaudioside A therein to directly produce rebaudioside D at a high density, without being affected by rebaudioside C in stevioside crude sugar, so it has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and genetic engineering, and specifically relates to a recombinant bacterium for catalyzing stevioside crude sugar to produce rebaudioside D and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Stevioside, also known as steviol glycoside, is a natural sugar-free sweetener extracted from the leaves of Stevia rebaudiana. The sweetness of stevioside is about 250-300 times that of sucrose, and it does not contain calories. It is known as the world's "third generation natural zero-calorie (zero-calorie) healthy sugar source". There are many types of stevioside compounds. At present, more than 30 types of stevioside compounds have been discovered, mainly including the more common stevioside (Stevioside, STV) and rebaudioside A (Rebaudioside A, RA), in addition to rebaudioside C (RC), rebaudioside D (RD), rebaudioside R (RR) and rebaudioside S (RS).

[0004] Among them, although rebaudioside D is not the main glycoside in stevia raw sugar, its bitterness and licorice aftertaste are significantly lower than rebaudioside A, and the toxicity studies of related structural substances have also confirmed the safety of rebaudioside D, which can be used in food, medicine and other fields. Therefore, rebaudioside D is a potential natural sweetener with better taste than rebaudioside A.

[0005] The traditional catalytic production method of rebaudioside D can only use rebaudioside A with higher purity as a substrate. The reason is that although stevioside crude sugar mainly contains stevioside and rebaudioside A, it also contains a certain amount of rebaudioside C (content is about 7%), and rebaudioside C has a certain inhibitory effect on the enzymatic activity of the original UDP-glucosyltransferase that catalyzes rebaudioside A to form rebaudioside D. Therefore, the efficiency of directly using stevioside crude sugar as a substrate to catalyze rebaudioside A to form rebaudioside D in the traditional way is relatively low. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a recombinant bacterium that catalyzes stevioside crude sugar to produce rebaudioside D and its application, which directly uses stevioside crude sugar as a substrate and catalyzes stevioside STV and rebaudioside A therein at a high density to directly produce rebaudioside D. Based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a recombinant bacterium, which at least expresses an ABC transporter, a glycosyltransferase and a sucrose synthase; wherein the expression of the ABC transporter is constitutive expression, the glycosyltransferases are UGT76G1 and UGTsl2mu, and the sucrose synthase is Atsus1. Specifically, the ABC transporter before the transformation will only start to express when it is under drug stress (i.e., inducible expression), which is not conducive to practical application, so it is transformed into constitutive expression.

[0009] In another specific embodiment of the present invention, the specific method for constitutively expressing an ABC transporter comprises: replacing the promoter of a gene encoding the ABC transporter with a constitutive promoter; wherein the amino acid sequence of the ABC transporter is as shown in SEQ ID NO.1-2.

[0010] The constitutive promoter may be PJ23119.

[0011] In another specific embodiment of the present invention, the amino acid sequence of the glycosyltransferase UGT76G1 is shown in SEQ ID NO.3, the amino acid sequence of the glycosyltransferase UGTsl2mu is shown in SEQ ID NO.4, and the amino acid sequence of the sucrose synthase Atsus1 is shown in SEQ ID NO.5.

[0012] In another specific embodiment of the present invention, the starting strain of the recombinant bacteria is Escherichia coli, further Escherichia coli BL21.

[0013] The second aspect of the present invention provides a method for constructing the above-mentioned recombinant bacteria, which comprises: using Escherichia coli as a starting strain, replacing the gene promoter encoding the ABC transporter with a constitutive promoter using gene editing technology to obtain a strain that constitutively expresses the ABC transporter; and transferring the encoding genes of exogenous glycosyltransferase and sucrose synthase into the strain that constitutively expresses the ABC transporter.

[0014] In another specific embodiment of the present invention, the gene editing technology may be CRISPR / Cas9 editing technology; specifically, the pEcCas / pEcgRNA editing system is used to implement gene editing of the starting strain Escherichia coli BL21; wherein, when the pEcCas / pEcgRNA editing system is used for gene editing, pTargetF-P is constructed. J23119 The nucleotide sequences of the amplification primers for the plasmid are shown in SEQ ID NO.6-7; the nucleotide sequence of the constructed donor DNA is shown in SEQ ID NO.8.

[0015] The specific method of transferring the coding genes of exogenous glycosyltransferase and sucrose synthase into the strain constitutively expressing ABC transporter comprises: constructing a first plasmid containing the coding gene of exogenous glycosyltransferase UGTSL2, and constructing a second plasmid containing exogenous glycosyltransferase UGT76G1 and sucrose synthase Atsus1; co-transforming the first plasmid and the second plasmid into the strain constitutively expressing ABC transporter;

[0016] Wherein, the nucleotide sequence of the exogenous glycosyltransferase UGTSL2 encoding gene is shown in SEQ ID NO.11;

[0017] The nucleotide sequences of the exogenous glycosyltransferase UGT76G1 and sucrose synthase Atsus1 are shown in SEQ ID NOs. 12-13, respectively.

[0018] The third aspect of the present invention provides the use of the above-mentioned recombinant bacteria in catalyzing the production of rebaudioside D from stevioside crude sugar.

[0019] A fourth aspect of the present invention provides a method for catalyzing stevioside crude sugar to produce rebaudioside D, the method comprising: adding the recombinant bacteria to a reaction system containing stevioside crude sugar to catalyze the production of rebaudioside D.

[0020] The reaction system containing stevia crude sugar specifically includes 100-300 g / L stevia crude sugar, 0.1-1 M sucrose, and 10-100 mM potassium dihydrogen phosphate. The pH of the reaction system is weakly alkaline, specifically 8.0. The specific conditions of the catalytic reaction are: catalytic reaction at 35-40° C. for 10-100 h, preferably catalytic reaction at 40° C. for 72 h.

[0021] Beneficial technical effects of the above technical solution:

[0022] 1) The above technical solution discovered the ABC transporter protein from Escherichia coli, which can effectively transport extracellular stevioside and rebaudioside A into the cell, and then transport the catalyzed rebaudioside D out of the cell. Since the transport efficiency of rebaudioside C by this protein is relatively low, the inhibitory effect of rebaudioside C contained in stevioside crude sugar on catalysis is reduced to a minimum during the entire catalytic transport process;

[0023] 2) The above technical solution can efficiently and directly catalyze high-density stevioside crude sugar after recombinant expression of three genes UGT76G1, Atusus and UGTsl2mu in Escherichia coli with constitutively expressed ABC transporter, and convert 2 / 3 of the total content of stevioside and rebaudioside A therein into rebaudioside D; experimental verification shows that the catalytic efficiency of Escherichia coli without constitutively expressed ABC transporter is only 1 / 4 of the catalytic efficiency of the bacteria, so the above technical solution has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 This is the plasmid map of pCDF-T7-UGTSL2 in the embodiment of the present invention.

[0026] Figure 2 This is the plasmid map of pCOLA-T7-UGT76G1-T7-Atsus1 in the examples of the present invention.

[0027] Figure 3 It is the liquid chromatogram of the substrate before catalysis in the embodiment of the present invention.

[0028] Figure 4 It is a liquid chromatogram after the conversion is completed in the embodiment of the present invention. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0031] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The pEcCas / pEcgRNA gene editing technology used in the examples can be found in the literature: Li, Q., Sun, B., Chen, J., Zhang, Y., Jiang, YU, & Yang, S. (2021). A modified pCas / pTargetF system for CRISPR-Cas9-assisted genomeediting in Escherichia coli. Acta Biochimica et Biophysica Sinica, 53 (5), 620-627.

[0032] Example

[0033] In this embodiment, Escherichia coli BL21 is used as the basic strain, and the gene promoters encoding HO397_02060 (SEQ ID NO.1) and HO397_02065 (SEQ ID NO.2) proteins are replaced with the constitutive promoter PJ23119 using the pEcCas / pEcgRNA gene editing technology; then, the Escherichia coli expression vectors pCOLAduet and pCDFduet are used to co-express Escherichia coli UGT76G1, Atsus1 and UGTsl2mu, and finally the co-expression plasmid is transformed into the modified Escherichia coli BL21, and the strain can achieve the high-density catalytic production of rebaudioside D by stevioside crude sugar.

[0034] ABC transporter sequence: 1.

[0036] (SEQ ID NO. 1) 2、

[0038] (SEQ ID NO.2)

[0039] UGT76G1 :ENKTETTVRRRRRIILFPPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEEVSCLITDALWYFAQSVADSNLLRRLVLMTSSLFNFH AHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSSSEVDEKDFLEIARGLVDSKQSF

[0040] LWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAF

[0041] WTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLEN

[0042] GWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL(SEQ ID NO.3)

[0043] UGTsl2mu

[0044] MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIP

[0045] EKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFS

[0046] RILQNLKPDLLIYDVLQPWAEHVANEQNIPAGKLLTSCAAVFSYFFSFRK

[0047] NPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTSRT

[0048] IEAKYIDYCTELCNWKVVPPVGPPFQDLITNDADNKELIDWLGTKHENST

[0049] VFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALPK

[0050] GFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSAMESIDFGVPII

[0051] AMPIHFDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAETLKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK(SEQ ID NO.4)

[0052] Atsus1

[0053] ANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIA

[0054] EFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVPRPRGVWEYL

[0055] RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPT

[0056] LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEK

[0057] IQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAER

[0058] VLDMIRLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLG

[0059] YPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTT

[0060] CGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAV

[0061] LOVELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDS

[0062] DIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYES

[0063] HTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSE

[0064] IEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNT

[0065] RLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWIS

[0066] SQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATC

[0067] KGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFFTKCKEDPSHWDEISKG

[0068] GLQRIEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDD(SEQID NO.5)

[0069] The specific construction method is as follows:

[0070] 1. Construction of strains constitutively expressing ABC-transporters:

[0071] The pEcCas / pEcgRNA gene editing system was used for gene editing. First, pTargetF-P J23119 Plasmids and donor DNA fragments.

[0072] Construction of pTargetF-P J23119 Plasmid: Primers P1 and P2 are annealed into double strands and then digested with BsaI endonuclease to obtain double-stranded DNA fragments with sticky ends at both ends. Then, the double-stranded DNA fragments are connected to the pTargetF linearized plasmid vector digested with BsaI using T4 DNA ligase. The ligation product is transformed into DH5α chemically competent cells and, after recovery, is applied to an LB plate containing spectinomycin (final concentration of 100 μg / ml). Single clones are selected for PCR verification and then verified by sequencing. If the verification is successful, the pTargetF-P J23119 Construction of plasmids.

[0073] Primer P1 sequence: ggtctcggttttGTTGTGTCCTGACCTGATTCTGG (SEQ ID NO.6)

[0074] Primer P2 sequence: CCAGAATCAGGTCAGGACACAACaaaaccgagacc (SEQ ID NO.7)

[0075] Prepare donor DNA fragments: The donor DNA consists of two homologous sequences and P J23119 The donor DNA is obtained by total gene synthesis, and its sequence is shown in SEQ ID NO.8.

[0076] cggggctgtcggacgtcacttccagcttcgcgatggaacagattaaatacaccacttctttacccatcgaataattgacagctagctcagtcctaggtataatgctagcgaattcattaaaga ggagaaaggtacagtgcgattatttgctcaattaagctggtatttccgtcgggaatggcgtcgctatctcggggctgtcgccttgcttgtcattatcgcgatgctgcaactggttccgcc(SEQ ID NO.8).

[0077] Gene editing process:

[0078] The plasmid pEcCas was transferred into the E. coli BL21 strain, and the BL21 / pEcCas clone was obtained on a plate containing kanamycin (final concentration of 50 μg / ml). The strain BL21 / pEcCas was inoculated with bacteria at a bacterial solution OD 600 When the value was 0.15-0.2, L-arabinose (final concentration was 20 mM) was added to induce the expression of the recombinase, and the culture was continued until the OD 600 When the value is 0.6-0.7, the culture is stopped and the bacterial solution is prepared into electroporation competent cells. At this time, the competent cells contain plasmid pEcCas and overexpressed recombinase. J23119 Co-transform the donor DNA into the competent cells, plate on LB plates containing kanamycin (final concentration of 50 μg / ml) and spectinomycin (final concentration of 100 μg / ml), and culture overnight at 30°C. Pick a single clone for PCR verification, and then verify it by sequencing. If the verification is successful, the gene editing is completed, and a recombinant engineering strain with constitutive expression of ABC-transporter is obtained. Among them, the sequence before replacement: atatccagaatcaggtcaggacacaacgc (SEQ ID NO.9); the sequence after replacement:

[0079] ttgacagctagctcagtcctaggtataatgctagcgaattcattaaagaggagaaaggtaca (SEQ IDNO.10)

[0080] 2. Plasmid construction:

[0081]

[0082] The optimal codon sequence corresponding to the UGT76G1 protein was optimized using E. coli codons:

[0083]

[0084] The optimal codon sequence corresponding to the Atsus1 protein was optimized using E. coli codons:

[0085]

[0086] atggcgcgtctggatcgtgttaaaaacctgagcggtctggtggaatggtacggcaagaacacccgtctgcgtgag

[0087] ctggcgaacctggtggttgtgggtggcgatcgtcgtaaagaaagcaaggacaacgaggaaaaagcggagatga

[0088] aaaagatgtacgacctgatcgaggaatataagctgaacggccagttccgttggattagcagccaaatggatcgtgt

[0089] gcgtaacggcgagctgtaccgttatatctgcgacaccaaaggcgcgttcgttcagccggcgctgtatgaagcgttt

[0090] ggtctgaccgttgttgaagcgatgacctgcggtctgccgacctttgcgacctgcaaaggtggcccggcggaaatc

[0091] attgtgcacggtaaaagcggctttcacattgatccgtaccacggcgaccaggcggcggataccctggcggacttc

[0092] tttaccaaatgcaaggaagatccgagccactgggacgagatcagcaaaggtggcctgcaacgtattgaggaaaa

[0093] gtacacctggcagatctatagccaacgtctgctgaccctgaccggtgtttatggcttctggaaacacgtgagcaac

[0094] ctggaccgtctggaagcgcgtcgttacctggagatgttttacgcgctgaagtatcgtccgctggcgcaggcggttccgctggcgcaagacgat(SEQ ID NO.13)

[0095] Primers were designed: p3: taataaggagatataccatggcaaccaatctgcgcg (SEQ ID NO.14), p4: ccagactcgagttatttgctcttattgctattgcgg (SEQ ID NO.15) to amplify the UGTSL2 gene, digested with NCOI+XbaI, and the E. coli expression vector pCDFduet was digested with NCOI+XbaI. After ligation with ligase, the plasmid was transformed into E. coli competent cells, positive clones were screened on streptomycin plates, and pCDF-UGTSL2 plasmid was identified by sequencing.

[0096] Primers were designed: p5: aataaggagatataccatggagaacaagaccgaaaccaccgtg (SEQ ID NO.16), P6: attatgcggccgcaagcttacaggctgctaatatagctaaccaggctttc (SEQ ID NO.17) to amplify UGT76G1, digested with NcoI+NotI, and the E. coli expression vector pCOLAduet was digested with NcoI+NotI. After ligation with ligase, the competent cells were transformed into E. coli, positive clones were screened on kanamycin plates, and pCOLA-UGT76G1 was identified by sequencing. P7: aggagatatacatatggcgaacgcggagcgtatg (SEQ ID NO.18), P8: ccagactcgagttaatcgtcttgcgccagcggaaccg (SEQ ID NO.19) AtSUS1 was amplified, digested with ndeI+xbaI, and pCOLA-UGT76G1 was digested with ndeI+xbaI. After ligation with ligase, it was transformed into E. coli competent cells, positive clones were screened on kanamycin plates, and pCOLA-UGT76G1-Atsus1 was identified by sequencing.

[0097] 3. Strain construction:

[0098] pCDF-UGTSL2; pCOLA-UGT76G1-Atsus1 were co-transformed into the BL21 strain that constitutively expresses ABC-transporter, and positive clones were screened under the dual resistance of kanamycin and streptomycin.

[0099] 4. Strain expression: The strain was cultured in TB medium until OD600 = 1, and IPTG was added to a final concentration of 1 mM. The culture was induced at 22°C overnight, and the cells were collected after centrifugation for later use.

[0100] 5. Catalysis: Prepare a catalytic solution of 50 mM potassium dihydrogen phosphate, 0.5 M sucrose, and 200 g / L stevioside crude sugar (RA 50%, STV 20% stevioside crude product) (pH 8.0). Add 200 g / L of bacterial cells, catalyze at 40°C for 72 hours until the catalysis is complete, and detect the catalytic results by liquid chromatography.

[0101] The RD was formed by catalysis of crude sugar, and the catalytic solution was prepared with 50 mM potassium dihydrogen phosphate, 0.5 M sucrose, and 100 g / L stevioside crude sugar (RA 50%, STV 20% stevioside crude product) (pH 8.0). 200 g / L of bacterial cells were added, and the catalysis was completed after catalysis at 40°C for 72 hours, and the catalytic results were detected by liquid chromatography.

[0102] The chromatogram of substrate before catalysis is as follows Figure 3 After the conversion is completed, the chromatogram is as shown in Figure 4 shown.

[0103] The RA content in the solution before catalysis is 50g / L, the STV content is 20g / L. After catalysis, the RD content in the solution is 33.5g / L, the RM content is 10.3g / L, and the stevioside substrate conversion rate can reach 100%.

[0104] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for catalyzing stevioside raw sugar to produce rebaudioside D, characterized in that: The method comprises: adding recombinant bacteria to a reaction system containing stevioside raw sugar to catalyze the production of rebaudioside D; The recombinant bacteria at least express ABC transporter, glycosyltransferase and sucrose synthase; wherein the expression mode of the ABC transporter is constitutive expression, the glycosyltransferases are UGT76G1 and UGTsl2mu, and the sucrose synthase is Atsus1; The amino acid sequence of the ABC transporter is shown in SEQ ID NO.1-2, the amino acid sequence of the glycosyltransferase UGT76G1 is shown in SEQ ID NO.3, the amino acid sequence of the glycosyltransferase UGTsl2mu is shown in SEQ ID NO.4, and the amino acid sequence of the sucrose synthase Atsus1 is shown in SEQ ID NO.5; The specific construction method of the recombinant bacteria includes: replacing the gene promoter encoding the ABC transporter with a constitutive promoter to obtain a strain constitutively expressing the ABC transporter; transferring the coding genes of exogenous glycosyltransferase and sucrose synthase into the strain constitutively expressing the ABC transporter; wherein the nucleotide sequence of the exogenous glycosyltransferase UGTSL2 coding gene is shown in SEQ ID NO.11; the nucleotide sequences of the exogenous glycosyltransferase UGT76G1 and sucrose synthase Atsus1 are shown in SEQ ID NO.12-13 respectively; The constitutive promoter is PJ23119; The starting strain of the recombinant bacteria is Escherichia coli BL21; The reaction system containing stevioside raw sugar specifically includes 100-300 g / L stevioside raw sugar, 0.1-1 M sucrose, and 10-100 mM potassium dihydrogen phosphate, and the pH of the reaction system is weakly alkaline; the specific conditions of the catalytic reaction are: catalytic reaction at 35-40° C. for 72-100 hours.

2. The method for catalyzing stevioside raw sugar to produce rebaudioside D according to claim 1, characterized in that: The construction method of the recombinant bacteria comprises: using Escherichia coli BL21 as a starting strain, replacing a gene promoter encoding an ABC transporter with a constitutive promoter by gene editing technology to obtain a strain constitutively expressing the ABC transporter; and transferring the coding genes of exogenous glycosyltransferase and sucrose synthase into the strain constitutively expressing the ABC transporter.

3. The method for catalyzing stevioside raw sugar to produce rebaudioside D as claimed in claim 2, characterized in that: The pEcCas / pEcgRNA editing system was used to achieve gene editing of the starting strain Escherichia coli BL21.

4. The method for catalyzing stevioside raw sugar to produce rebaudioside D as claimed in claim 3, characterized in that: The specific method of transferring the coding genes of exogenous glycosyltransferase and sucrose synthase into the strain constitutively expressing ABC transporter comprises: constructing a first plasmid containing the coding gene of exogenous glycosyltransferase UGTSL2, and constructing a second plasmid containing exogenous glycosyltransferase UGT76G1 and sucrose synthase Atsus1; co-transforming the first plasmid and the second plasmid into the strain constitutively expressing ABC transporter; Wherein, the nucleotide sequence of the exogenous glycosyltransferase UGTSL2 encoding gene is shown in SEQ ID NO.11; The nucleotide sequences of the exogenous glycosyltransferase UGT76G1 and sucrose synthase Atsus1 are shown in SEQ ID NO.12-13 respectively.

5. Use of the method of catalyzing stevioside crude sugar to produce rebaudioside D according to claim 1 in catalyzing stevioside crude sugar to produce rebaudioside D.

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