Xanthosine acid producing strain, construction method and application thereof

Abstract

The application provides a xanthosine production strain and a construction method and application thereof. The strain is constructed by knocking out E. coli on the W3110 genome surE , ushA, nagD , blocking the xanthosine decomposition pathway; knocking out purR genes, removing the feedback inhibition of PurR protein on the IMP synthesis pathway; integrating an artificial operon PUR to synergistically strengthen the IMP de novo synthesis pathway; replacing the natural promoter of fliC with a promoter P guaA , selectively regulating the synthesis of guanosine to reduce the consumption of xanthosine while maintaining the growth of the bacterial body; overexpressing , prs D128A , guaB , strengthening the expression of key enzymes in the xanthosine synthesis pathway, enhancing the xanthosine synthesis capacity; overexpressing hpt, gpt , enhancing the xanthine replenishment to xanthosine, further enhancing the xanthosine synthesis metabolic pathway, and stably and efficiently producing xanthosine.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and fermentation engineering technology, and in particular to a xanthocyanidin-producing strain, its construction method, and its application. Background Technology

[0002] Xanthosine monophosphate (XMP), also known as xanthosine ribonucleotide, is a key intermediate in the purine nucleotide metabolic pathway and a precursor in the biosynthesis of guanylic acid (GMP) and its derivatives. XMP can be used as a flavor enhancer in food additives to improve flavor, and it also serves as an intermediate in the synthesis of antiviral drugs and immunomodulators. In recent years, with the development of biomanufacturing technology, the application potential of XMP has been further expanded. However, its production cost and efficiency remain key issues restricting large-scale application. Therefore, developing economical and efficient synthetic methods is of significant practical importance for production.

[0003] Currently, the main methods for producing flavin glycosides include chemical synthesis, enzymatic catalysis, and microbial fermentation. The commonly used microbial fermentation strain is *Corynebacterium ammoniagenes*, but its genetic background modification is not precise or systematic enough. Furthermore, *Corynebacterium ammoniagenes* has a slow growth rate and a relatively long fermentation cycle. *Escherichia coli*, as a commonly used chassis cell for metabolic engineering, has the advantages of a clear genetic background, ease of modification, rapid growth, and a short fermentation cycle. Microbial fermentation utilizes engineered strains to synthesize flavin glycosides de novo through metabolic pathways, using inexpensive carbon sources such as glucose as raw materials, offering advantages such as low cost, mild conditions, and environmental friendliness. Therefore, systematically constructing *E. coli* engineered strains capable of efficiently synthesizing flavin glycosides using metabolic engineering techniques is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain for producing xanthocyanidins.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned xanthic acid producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned xanthic acid producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A strain that produces flavin glycosides, named strain E.coli Xmp11 It is the starting strain E.coli It was obtained from W3110 through targeted gene modification, specifically by knocking out the 5' / 3'-nucleotidase gene. surE 5'-nucleotidase gene ushA 5'-nucleotidase gene nagDand purine synthesis repressor protein gene purR Overexpression of phosphoribosyl pyrophosphate synthase gene prs D128A , hpt, gpt and guaB Heterogeneous introduction from Bacillus subtilis Amide phosphoribosyltransferase gene Using the E. coli flagellar filament structural protein gene fliC Promoter P of (Gene ID: 949101) fliC Replace GMP synthase gene guaA natural promoter (P) pyrH ); Integrate the artificial manipulator PUR.

[0008] Preferably, in the above-mentioned xanthic acid-producing strain, the directional modification method utilizes CRISPR / Cas9 gene editing technology to completely modify the starting strain. E.coli Modifications were made to the genome of chromosome W3110.

[0009] Preferably, the above-mentioned xanthinolic acid producing strain, the starting strain is E.coli W3110 ATCC 27325.

[0010] Preferably, in the above-mentioned xanthinolic acid producing strain, the artificial operon PUR is... prs D128A , , purD, purM, purN, purT, purL, purE, purK, purC, purB bsu and purH bsu The genes were sequentially linked using RBS, and the nucleotide sequence of the artificial operon PUR is shown in SEQ ID NO.11 of the sequence listing.

[0011] Preferably, the above-mentioned xanthinolic acid producing strain, wherein prs D128A The nucleotide sequence of (Gene ID: 945772) is shown in SEQ ID NO. 7 of the sequence listing; The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing; purD The nucleotide sequence of (Gene ID: 948504) is shown in SEQ ID NO. 16 of the sequence listing; purN The nucleotide sequence of (Gene ID: 946973) is shown in SEQ ID NO. 17 of the sequence listing; purT The nucleotide sequence of (Gene ID: 946368) is shown in SEQ ID NO. 18 of the sequence listing; purLThe nucleotide sequence of (Gene ID: 947032) is shown in SEQ ID NO. 19 of the sequence listing; purM The nucleotide sequence of (Gene ID: 946975) is shown in SEQ ID NO. 20 of the sequence listing; purK The nucleotide sequence of (Gene ID: 945153) is shown in SEQ ID NO. 21 of the sequence listing; purE The nucleotide sequence of (Gene ID: 949031) is shown in SEQ ID NO. 22 of the sequence listing; purC The nucleotide sequence of (Gene ID: 946957) is shown in SEQ ID NO. 23 of the sequence listing; purB bsu The nucleotide sequence of (Gene ID: 936048) is shown in SEQ ID NO. 24 of the sequence listing; purH bsu The nucleotide sequence of (GeneID:936053) is shown in SEQ ID NO.25 of the sequence listing.

[0012] The above prs D128A , , purD, purM, purN, purT, purL, purE, purK, purC The gene originated from E. coli Escherichia coli ; purB bsu and purH bsu The gene originates from Bacillus subtilis Bacillus subtilis .

[0013] Preferably, the nucleotide sequence of the above-mentioned xanthinolic acid producing strain, wherein the RBS is shown in SEQ ID NO.26.

[0014] Preferably, the above-mentioned xanthinolic acid producing strains have a genome... gapC Site-integrated from B.subtilis 168 amide phosphoribosyltransferase gene Using the promoter P trc Regulation; in the genome yjgX Integrating phosphoribosyl pyrophosphate synthase gene at the site prs D128A Using the promoter P trc Regulation; in the genome mbhA IMP dehydrogenase gene integrated at the site guaB Using the promoter P T7 Regulation; in the genome ylbE Integrating hypoxanthine phosphoribosyltransferase gene at the site hpt Using the promoter P trcRegulation; in the genome yncI Integrating guanine hypoxanthine phosphoribosyltransferase gene at the site gpt Using the promoter P trc Regulation; in the genome ilvG In the de novo synthesis module of IMP (inosine monophosphate) segmental integration at the site prs D128A , , purD, purM, purN, purT, purL, purE, purK, purC, purB bsu and purH bsu Twelve genes are sequentially linked using RBS to form the artificial operon PUR, sharing a common set of promoters P. trc Regulation.

[0015] Preferably, the above-mentioned xanthinolic acid producing strain, wherein surE The nucleotide sequence of (Gene ID: 947211) is shown in SEQ ID NO. 1 of the sequence listing. ushA The nucleotide sequence of (Gene ID: 947331) is shown in SEQ ID NO. 2 of the sequence listing. nagD The nucleotide sequence of (Gene ID: 945283) is shown in SEQ ID NO. 3 of the sequence listing. purR The nucleotide sequence of (Gene ID: 945226) is shown in SEQ ID NO. 4 of the sequence listing.

[0016] Preferably, the above-mentioned xanthinolic acid producing strain, wherein guaA The nucleotide sequence of (Gene ID: 947334) is shown in SEQ ID NO. 5 of the sequence listing. guaB The nucleotide sequence of (Gene ID: 946985) is shown in SEQ ID NO. 8 of the sequence listing. hpt The nucleotide sequence of (Gene ID: 946624) is shown in SEQ ID NO. 9 of the sequence listing. gpt The nucleotide sequence of (Gene ID: 944817) is shown in SEQ ID NO. 10 of the sequence listing.

[0017] Preferably, the above-mentioned xanthinolic acid producing strain, wherein P trc The nucleotide sequence of the promoter is shown in SEQ ID NO. 12 of the sequence listing; P T7 The nucleotide sequence of the promoter is shown in SEQ ID NO.13 of the sequence listing; promoter P fliC The nucleotide sequence is shown in SEQ ID NO. 14 of the sequence listing; guaA natural promoter (P) pyrHThe nucleotide sequence of is shown in the sequence listing SEQ ID NO.15.

[0018] The specific steps for constructing the above-mentioned xanthinolic acid-producing strain are as follows: (1) In E.coli Knockout of strain W3110 surE , ushA, nagD Blocking the xanthoside degradation pathway; knocking out purR Gene, to relieve the feedback inhibition of the IMP synthesis pathway by the PurR protein; (2) Through the genome gapC and yjgX Overexpression at each site , prs D128A and with P trc Promoters regulate separately and in the genome mbhA Overexpression at site guaB, and use P T7 Promoter regulation enhances the expression of key enzymes in the xanthocyanidin synthesis pathway; (3) Through the genome ylbE, yncI Overexpression at each site hpt, gpt and with P trc Promoter regulation enhances xanthine replenishment and the generation of xanthocyanidins; (4) Using the E. coli flagellar filament structural protein gene fliC promoter P fliC Replace the GMP synthase gene in the genome guaA The natural promoter selectively and tunably regulates the synthesis of guanylic acid, thereby reducing the consumption of xanthocyanin acid while maintaining bacterial growth. (5) In ilvG Segmented integration of artificial operon PUR at site prs D128A , , purD, purM, purN, purT, purL, purE, purK, purC, purB bsu and purH bsu A total of 12 genes were sequentially linked using RBS, and P was used to connect them. trc Promoter regulation synergistically enhances the IMP de novo synthesis pathway.

[0019] Applications of the above-mentioned xanthinolic acid producing strains in the production of xanthinolic acid.

[0020] Preferably, the above-mentioned application involves the production of xanthocyanidins through fermentation.

[0021] Preferably, in the above application, xanthocyanidins are obtained by shake-flask culture, and the specific steps are as follows: (1) Activation of bacterial strain: Inoculate the bacterial solution from the preservation tube into the slant medium for activation culture; transfer the colonies from the slant medium to LB medium for further activation culture; (2) Inoculate 10%-15% into the fermentation medium, seal and shake to culture. During fermentation, maintain pH at 7.0-7.2 by adding ammonia water; add glucose solution to maintain fermentation.

[0022] Preferably, in the above application, the slant culture medium used in step (1) is: glucose 2g / L, peptone 10g / L, yeast extract 5g / L, sodium chloride 2.5g / L, KH2PO4 1.0g / L, MgSO4·7H2O 0.2g / L, agar powder 25%, the remainder being water, pH 7.0-7.2; the LB culture medium used is: yeast extract 5g / L, peptone 10g / L, NaCl 10g / L, agar powder 20g / L, the remainder being water.

[0023] Preferably, in the above application, the fermentation culture medium used in step (2) is: monosodium glutamate 3.5 g / L, yeast powder 8 g / L, citric acid 2 g / L, methionine 0.3 g / L, sodium hexametaphosphate 2 g / L, ammonium sulfate 3 g / L, MgSO4·7H2O 3 g / L, KH2PO4 4 g / L, FeSO4·7H2O 25 mg / L, MnSO4 10 mg / L, V B1 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, phenol red 2%, the remainder is water.

[0024] Preferably, in the above application, flavin is produced by fermentation in a fermenter, and the specific steps are as follows: 1) Seed activation: After the inoculum is evenly spread on the activation slant and cultured, it is transferred to a flask for further culture; 2) Seed culture: The bacterial suspension was inoculated into the seed culture medium and cultured at pH 7.0, constant temperature of 36℃, and dissolved oxygen of 30%-60%. 3) Fermentation culture: Waiting for the OD of the seed cell count 600 At 20-25°C, inoculate the fermentation medium at a rate of 20% to start fermentation. During fermentation, control the pH at 7.0, the temperature at 36°C, and the dissolved oxygen at 30%-60%. After the glucose in the medium is consumed, add glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L.

[0025] Preferably, in the above application, the slant culture medium used in step 1) is: glucose 2g / L, peptone 10g / L, yeast powder 5g / L, sodium chloride 2.5g / L, KH2PO4 1.0g / L, MgSO4·7H2O 0.2g / L, agar powder 25%, the remainder being water, pH 7.0-7.2.

[0026] Preferably, in the above application, the seed culture medium used in step 2) is: glucose 10 g / L, yeast extract 8 g / L, peptone 2 g / L, ammonium sulfate 2 g / L, KH2PO4 3 g / L, methionine 0.3 g / L, V B1 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 1 g / L, the remainder is water.

[0027] Preferably, in the above application, the fermentation medium used in step 3) is: glucose 10g / L, yeast extract 8g / L, citric acid 5g / L, ammonium sulfate 3g / L, KH2PO4 6g / L, MgSO4·7H2O 6g / L, sodium hexametaphosphate 2g / L, betaine 5g / L, FeSO4·7H2O 25mg / L, MnSO4 10mg / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 2 mg / L, the remainder is water.

[0028] All of the above-mentioned culture media can be prepared using standard methods.

[0029] Beneficial effects: The above-mentioned xanthic acid-producing strains were used in CRIPSR / Cas9 gene editing technology to... E.coli On the W3110 genome, the first knockout surE , ushA, nagD Blocking the xanthoside degradation pathway; knocking out purR Genes were used to relieve the feedback inhibition of the IMP synthesis pathway by the PurR protein; the artificial operon PUR was integrated to synergistically enhance the de novo IMP synthesis pathway; and [other methods were employed]. fliC promoter P fliC Replacement genome guaA The natural promoter selectively and tunably regulates guanylic acid synthesis, reducing guanylic acid consumption while maintaining bacterial growth; overexpression , prs D128A , guaBThis enhanced the expression of key enzymes in the xanthocyanin synthesis pathway, thereby increasing the synthesis capacity of xanthocyanin; finally, through overexpression... hpt, gpt This study enhanced the conversion of xanthine to xanthoside A, further strengthening the xanthoside A biosynthetic pathway. The obtained strain is plasmid-free, defect-free, requires no induction, and possesses advantages such as good genetic stability, high fermentation yield, and low production cost. It is an excellent strain for stable and efficient production of xanthoside A, realizing a de novo synthesis of xanthoside A from glucose. After 32 hours of shake-flask fermentation, the xanthoside A yield reached as high as 3.5 g / L, and after 48 hours of fermenter fermentation, the yield reached 20.4 g / L, demonstrating broad application prospects. Attached Figure Description

[0030] Figure 1 Xanthocyanidin-producing strain E.coli Xmp11 A diagram illustrating the construction method. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0032] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 ml. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0033] The starting strain used in the examples was wild-type Escherichia coli. E.coli W3110 ATCC 27325 (commercially available); Wild-type Bacillus subtilis Bacillus subtilis 168 ATCC 23857 (commercially available); the artificial manipulator PUR gene sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd.; the corresponding promoter and gene of the genome are shown in the sequence listing.

[0034] The gene editing method used is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli Using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.), the engineered plasmid pGRB involved in this method uses pUC18 as its backbone and includes the promoter J23100, the gRNA-Cas9 binding region sequence, the terminator sequence, and ampicillin resistance (working concentration: 100 mg / L). The technical terms related to gene integration and plasmid construction used in the following examples are explained in the article. Primers used in the strain construction process are shown in Table 1.

[0035] Table 1 Primers used in strain construction

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] Example 1 like Figure 1 As shown, flavin acid engineered strain E.coli The method for constructing Xmp11 is as follows: The starting strain... E.coli W3110 was prepared according to the operation mentioned in CN202210417923.1. E.coli W3110 / pRed-Cas9 electrotransduction competent cells.

[0044] 1.1 Genes surE Knockout DNA fragments annealed using pGRB-surE-s and pGRB-surE-a primers were ligated to plasmid pGRB to construct the pGRB-surE plasmid. Then, primers surE-1, surE-2, surE-3, and surE-4 were used to ligate the DNA fragments. E.coli Using the genome of W3110 as a template, the upstream homologous arm surE-up and the downstream homologous arm surE-dw were obtained; then the fusion fragment was obtained. surE. Finally, the pGRB-surE plasmid was combined with the overlapping fragment. surE electric transfer to E.coli The strain W3110 was obtained by electrocompetent cell transfer; positive transformants were screened using surE-1 and surE-4 primers to obtain the strain. E.coli Xmp1.

[0045] 1.2 Genes ushA Knockout DNA fragments annealed using pGRB-ushA-s and pGRB-ushA-a primers were ligated to plasmid pGRB to construct the pGRB-ushA plasmid. Then, primers ushA-1, ushA-2, ushA-3, and ushA-4 were used to ligate the DNA fragments. E.coli Using the genome of W3110 as a template, the upstream homologous arm ushA-up and the downstream homologous arm ushA-dw were obtained; then the fusion fragment was obtained. ushA. Finally, the pGRB-ushA plasmid was combined with the overlapping fragment. ushA electricity transfer to E.coli Xmp1 was electrocompetent in cells; positive transformants were screened using ushA-1 and ushA-4 primers to obtain the strain. E.coli Xmp2.

[0046] 1.3 Genes nagD Knockout DNA fragments annealed using pGRB-nagD-s and pGRB-nagD-a primers were ligated to plasmid pGRB to construct the pGRB-nagD plasmid. Then, using nagD-1, nagD-2, nagD-3, and nagD-4 primers... E.coli Using the W3110 genome as a template, the upstream homologous arm nagD-up and the downstream homologous arm nagD-dw were obtained; then the fusion fragment was obtained. nagD. Finally, the pGRB-nagD plasmid was combined with the overlapping fragment. nagD electricity transfer to E.coli Xmp2 was electrocompetent in cells; positive transformants were screened using nagD-1 and nagD-4 primers to obtain the strain. E.coli Xmp3.

[0047] 1.4 Genes purR Knockout DNA fragments annealed using pGRB-purR-S and pGRB-purR-A primers were ligated to plasmid pGRB to construct the pGRB-purR plasmid. Then, using purR-QC-1, purR-QC-2, purR-QC-3, and purR-QC-4 primers, the DNA fragments were ligated to the pGRB plasmid. E.coli Using the W3110 genome as a template, the upstream homologous arm purR-up and the downstream homologous arm purR-dw were obtained; then the fusion fragment was obtained. purR. Finally, the pGRB-purR plasmid was combined with the overlapping fragment. purR electric transfer to E.coliXmp3 was electrocompetent in cells; positive transformants were screened using purR-QC-1 and purR-QC-4 primers to obtain the strain. E.coli Xmp4.

[0048] 1.5 Integration of the artificial manipulator PUR ( ilvG ::P trc -PUR integration) Using the *E. coli* W3110 genome as a template, primers ilvG-1 and ilvG-trc-2, ilvG-trc-3, ilvG-4#-3, and ilvG-1#-3 were used with ilvG-4 to obtain the upper homologous arm ilvG-trc-UP and the lower homologous arms ilvG-trc-DW, ilvG-4#-DW, and ilvG-1#-DW, respectively. Using the artificial operon PUR synthesized by Witty Gene as a template, primer PUR-①-TR was used. PCR amplification was performed using C-S and PUR-①-4#-A, PUR-②-S and PUR-②-1#-A, and PUR-③-S and PUR-③-trc-A to obtain the target fragments PUR-①, PUR-②, and PUR-③. Using the recovered upstream and downstream homologous arms ilvG-1 and ilvG-4#-DW and the target fragment PUR-① as templates, overlap PCR was performed with primers ilvG-1 and ilvG-4 to obtain the target fragment ilvG-P required for integration. trc Following the initial step of annealing primers pGRB-ilvG-S and pGRB-ilvG-A, the resulting DNA fragment was ligated to plasmid pGRB to construct the pGRB-ilvG plasmid. Finally, the purified ilvG-P... trc -PUR-① The integration fragment and plasmid pGRB-ilvG were simultaneously transferred via electroporation. E.coli In Xmp4 electrocompetent cells, positive transformants were screened using primers ilvG-1 and ilvG-4 for identification, and the strain was finally obtained. E.coli Xmp5-1; Using PUR-② and ilvG-1#-DW as templates, the target fragment ilvG-1#-PUR-② was obtained by overlap PCR with primers PUR-②-S and ilvG-4. Then, the DNA fragment obtained by annealing primers pGRB-4#-S and pGRB-4#-A was ligated to plasmid pGRB to construct plasmid pGRB-4#. Finally, the purified ilvG-1#-PUR-② integration fragment and plasmid pGRB-4# were simultaneously transformed into pGRB-4# by electroporation. E.coli In Xmp5-1 electrocompetent cells, positive transformants were screened using primers PUR-②-S and ilvG-4 for identification, and the strain was finally obtained. E.coli Xmp5-2; Using PUR-③ and ilvG-trc-DW as templates, the target fragment ilvG-P required for integration was obtained by overlap PCR using primers PUR-③-S and ilvG-4. trc Following step -PUR-③, the DNA fragment obtained by annealing primers pGRB-1#-S and pGRB-1#-A was ligated to plasmid pGRB to construct plasmid pGRB-1#. Finally, the purified ilvG-P trc -PUR-③ The integration fragment and plasmid pGRB-1# were simultaneously transferred via electroporation. E.coli In Xmp5-2 electrocompetent cells, positive transformants were screened using primers PUR-③-S and ilvG-4 to finally obtain the strain. E.coli Xmp5-3.

[0049] 1.6 genes guaA Replace the starter (P) pyrH ::P fliC ) Using *E. coli* W3110 as a template, PCR amplification was performed using primers UP-PfliC-s and UP-guaA-PfliC-A, guaA-PfliC-DW-S and guaB-A to obtain the upper homologous arm UP-PguaA-fliC and the lower homologous arm DW-PguaA-A. Using the recovered upper and lower homologous arms as templates, overlap PCR was performed using primers UP-fliC-s and guaB-A to obtain the target fragment PguaA-fliC for integration. Then, the DNA fragment obtained by annealing primers pGRB-PguaA-s and pGRB-PguaA-a was ligated to plasmid pGRB to construct the pGRB-PguaA plasmid. Finally, the purified PguaA-fliC integration fragment and plasmid pGRB-PguaA were simultaneously transformed into the target fragment by electroporation. E.coli In Xmp5-3 electrocompetent cells, positive transformants were screened using primers UP-fliC-s and guaB-A to finally obtain the strain. E.coli Xmp6.

[0050] 1.7 genes integration DNA fragments annealed using pGRB-gapC-s and pGRB-gapC-a primers were ligated to plasmid pGRB to construct the pGRB-gapC plasmid. Then, primers gapC-US, gapC-UA, gapC-DS, gapC-DA, purF-1, purF-2, purF-3, purF-4, purF-5, and purF-6 were used to ligate the DNA fragments. B. subtilis 168 and E.coliUsing the W3110 genome as a template, the upstream homologous arm gapC-up, the downstream homologous arm gapC-dw, and the target gene fragments purF1, purF2, and purF3 were obtained; the overlapping fragments purF1, purF2, and purF3 were used to obtain the target gene fragment purF; then, the fusion fragment gapC::purF was obtained. Finally, the pGRB-gapC plasmid and the overlapping fragment gapC::purF were electroporated to... E.coli Xmp6 was electrocompetent in cells; positive transformants were screened using gapC-US and gapC-DA primers to obtain the strain. E.coli Xmp7.

[0051] 1.8 genes prs D128A integration DNA fragments annealed using pGRB-yjgX-s and pGRB-yjgX-a primers were ligated to plasmid pGRB to construct the pGRB-yjgX plasmid. Then, using primers yjgX-US, yjgX-UA, prsA-s, prsA-a, yjgX-DS, and yjgX-DA, the DNA fragments were ligated to pGRB-yjgX-s and pGRB-yjgX-a. E.coli Using the genome of W3110 as a template, the upstream homologous arm yjgX-up and the downstream homologous arm yjgX-dw were obtained, along with the target gene. prs D128A Then obtain the fusion fragment yjgX:: prs D128A Finally, the pGRB-yjgX plasmid was combined with the overlapping fragment yjgX:: prs D128A Electric transfer to E.coli Xmp7 was electrocompetent in cells; positive transformants were screened using yjgX-US and yjgX-DA primers to obtain the strain. E.coli Xmp8.

[0052] 1.9 genes guaB integration DNA fragments annealed using pGRB-mbhA-S and pGRB-mbhA-A primers were ligated to plasmid pGRB to construct the pGRB-mbhA plasmid. Then, primers mbhA-1, mbhA-2, guaB-S, guaB-A, mbhA-3, and mbhA-4 were used to ligate the DNA fragments. E.coli Using the W3110 genome as a template, the upstream homologous arm mbhA-up, the downstream homologous arm mbhA-dw, and the target gene guaB were obtained; then, the fusion fragment mbhA::guaB was obtained. Finally, the pGRB-mbhA plasmid and the overlapping fragment mbhA::guaB were electroporated into... E.coliXmp8 was electrocompetent in cells; positive transformants were screened using mbhA-1 and mbhA-4 primers to obtain the strain. E.coli Xmp9.

[0053] 1.10 genes hpt integration DNA fragments annealed using pGRB-ylbE-s and pGRB-ylbE-a primers were ligated to plasmid pGRB to construct the pGRB-ylbE plasmid. Then, primers ylbE-up-s, ylbE-up-a, hpt-trc-S, hpt-A, ylbE-dw-s, and ylbE-dw-a were used to ligate the DNA fragments. E.coli Using the W3110 genome as a template, the upstream homologous arm ylbE-up, the downstream homologous arm ylbE-dw, and the target gene hpt were obtained; then, the fusion fragment ylbE::hpt was obtained. Finally, the pGRB-ylbE plasmid and the overlapping fragment ylbE::hpt were electroporated to... E.coli Xmp9 was electrocompetent in cells; positive transformants were screened using ylbE-up-s and ylbE-dw-a as identification primers to obtain the strain. E.coli Xmp10.

[0054] 1.11 genes gpt integration DNA fragments annealed using pGRB-yncI-s and pGRB-yncI-a primers were ligated to plasmid pGRB to construct the pGRB-yncI plasmid. Primers yncI-US, yncI-UA, trc-gpt-S, trc-gpt-A, yncI-DS, and yncI-DA were used to ligate the DNA fragments. E.coli Using the W3110 genome as a template, the upstream homologous arm yncI-up, the downstream homologous arm yncI-dw, and the target gene gpt were obtained; then, the fusion fragment yncI::gpt was obtained. Finally, the pGRB-yncI plasmid and the overlapping fragment yncI::gpt were electroporated to... E.coli Xmp10 was electrocompetent in cells; positive transformants were screened using yncI-US and yncI-DA primers to obtain the strain. E.coli Xmp11.

[0055] Example 2 The strain obtained in Example 1 E.coli Xmp11 is fermented in a shake flask to produce xanthic acid. The specific steps are as follows: 2.1 Culture medium 2.1.1 Slant Culture Medium Glucose 2g / L, peptone 10g / L, yeast powder 5g / L, sodium chloride 2.5g / L, KH2PO4 1.0g / L, MgSO4·7H2O 0.2g / L, agar powder 25%, dissolved in water, then adjusted to pH 7.0-7.2 with sodium hydroxide, and brought to a final volume of 500ml. The solution was dispensed into test tubes (9ml / tube) and flasks (45ml / flask), and sterilized in a high-pressure steam autoclave at 121℃ for 20min.

[0056] 2.1.2 LB medium Yeast powder 5g / L, peptone 10g / L, NaCl 10g / L, agar powder 20g / L, the remainder is water.

[0057] 2.1.3 Fermentation medium Monosodium glutamate 3.5 g / L, yeast powder 8 g / L, citric acid 2 g / L, methionine 0.3 g / L, sodium hexametaphosphate 2 g / L, ammonium sulfate 3 g / L, MgSO4·7H2O 3 g / L, KH2PO4 4 g / L, FeSO4·7H2O 25 mg / L, MnSO4 10 mg / L, V B1 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, phenol red 2%, the remainder is water.

[0058] 2.2 Cultivation Methods 2.2.1 Seed activation and culture: Inoculate the bacterial culture from the preservation tube into an agar slant and activate it for 12 hours; then transfer the colonies from the agar slant to LB medium and continue activation and culture for 12 hours at a temperature of 32°C.

[0059] 2.2.2 Fermentation culture: Inoculate 10%-15% of the culture medium into the fermentation medium, seal with nine layers of gauze, and incubate at 36℃ with shaking at 240 rpm. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia. Add 60% glucose solution to maintain fermentation for 30-32 hours. (Use phenol red as an indicator; when the fermentation broth color no longer changes, it indicates a sugar deficiency; add 1-2 ml of 60% (m / v) glucose solution if a sugar deficiency occurs). The fermentation cycle is 32 hours. No antibiotics or inducers are added during fermentation.

[0060] After 32 hours of shake-flask fermentation, the yield of xanthocyanidins can reach up to 3.5 g / L.

[0061] Example 3 Using the xanthic acid producing strain described in Example 1 E.coliXmp4 to Xmp11 were fermented in shake flasks for verification. The shake flask fermentation method is described in Example 2.

[0062] 3.1 Comparison of artificial manipulator PUR and manipulator pur ( EKBCSQLFMNHD The effect of the manipulator pur ( EKBCSQLFMNHD The operon gene in CN117229987A (A genetically engineered bacterium that produces hypoxanthine and its construction method and application) pur ( EKBCSQLFMNHD The integration method refers to the integration of the artificial manipulator PUR in Example 1, 1.5, as shown in Table 2.

[0063] Table 2 Strain type OD Xanthosine monophosphate yield (g / L) E.coli Xmp4 46.8 0.53 E. coli Xmp5-3 43.7 0.75 E.coli Xmp4+operator() 42.6 0.61

[0064] The results of the shake-flask fermentation above show that the strain with the self-constructed artificial operon PUR has a higher flavin yield (0.75 g / L) and a better growth status. Therefore, this strain was selected as E. coli Xmp5-3 for further modification.

[0065] 3.2 Contrast Knockout guaA Replacement guaA The effects of the promoter are shown in Table 3.

[0066] Table 3 strains OD Xanthocyanidin yield (g / L) E. coli Xmp5-3 43.7 0.75 E.coli Xmp5-3-Δ 25.9 0.80 E.coli Xmp5-3 promoter replacement for natural promoter 37.6 0.89

[0067] The results of the shake-flask fermentation described above show that, with fliC Promoter replacement guaA The strain with the natural promoter has a higher flavin yield (0.89 g / L) and does not require the addition of defective substances; therefore, this strain was selected as the [selected strain]. E. coli Xmp6 underwent further modifications.

[0068] 3.3 Comparison of subsequent fermentation experiment data for gene overexpression is shown in Table 4.

[0069] Table 4

[0070] The above shake-flask fermentation results revealed that strains with enhanced flavoside recombinant pathways... E. coli Xmp11 has the highest flavin yield, at 3.50 g / L.

[0071] Example 4 Using the xanthic acid production strain described in Example 1 E. coli Xmp11 was used to produce xanthic acid in a 5L fermenter.

[0072] 4.1 Culture medium 4.1.1 Slant Culture Medium Glucose 2g / L, peptone 10g / L, yeast powder 5g / L, sodium chloride 2.5g / L, KH2PO4 1.0g / L, MgSO4·7H2O 0.2g / L, agar powder 25%, dissolved in water, then adjusted to pH 7.0-7.2 with sodium hydroxide, and brought to a final volume of 500ml. The solution was dispensed into test tubes (9ml / tube) and flasks (45ml / flask), and sterilized in a high-pressure steam autoclave at 121℃ for 20min.

[0073] 4.1.2 Seed Culture Medium Glucose 10g / L, yeast extract 8g / L, peptone 2g / L, ammonium sulfate 2g / L, KH2PO4 3g / L, methionine 0.3g / L, vitamin C B1 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 1 g / L, the remainder is water.

[0074] 4.1.3 Fermentation medium Glucose 10g / L, yeast powder 8g / L, citric acid 5g / L, ammonium sulfate 3g / L, KH2PO4 6g / L, MgSO4·7H2O 6g / L, sodium hexametaphosphate 2g / L, betaine 5g / L, FeSO4·7H2O 25mg / L, MnSO4 10mg / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 2 mg / L, the remainder is water.

[0075] 4.2 Cultivation Methods 4.2.1 Seed activation: The inoculum was evenly spread on the activation slant and incubated at 37℃ for 12 h, then transferred to a flask and incubated for another 12 h; 4.2.2 Seed culture: The bacterial suspension was inoculated into the seed culture medium, pH 7.0, temperature constant at 36℃, dissolved oxygen at 30%-60%, and cultured for 7.5h; 4.2.3 Fermentation culture: waiting for the seed cell count OD 600 At 20-25°C, inoculate the fermentation medium at a rate of 20% to begin fermentation. During fermentation, maintain pH 7.0, temperature 36°C, and dissolved oxygen at 30%-60%. Once the glucose in the medium is depleted, add an 80% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L. Do not add antibiotics or inducers during fermentation.

[0076] After 48 hours of fermentation in a fermenter, the yield of xanthocyanidins can reach up to 20.4 g / L.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A xanthic acid-producing strain, characterized in that: The starting strain E. coli obtained by gene directed evolution method based on W3110, specifically, knocking out 5' / 3'-nucleotidase gene surE , 5'-nucleotidase gene ushA , 5'-nucleotidase gene nagD and purine synthesis repressor gene purR ; overexpressing phosphoribosyl pyrophosphate synthetase gene prs D128A , hpt and gpt regulated by promoter P trc ; Overexpression of phosphoribosyl pyrophosphate synthase gene guaB Using the promoter P T7 Regulation; introduction of heterogeneous sources Bacillus subtilis Amide phosphoribosyltransferase gene purF * bsu Using the promoter P trc Regulation; using E. coli flagellar filament structural protein genes fliC promoter P fliC Replace GMP synthase gene guaA The natural promoter; integrating the artificial operator PUR, using the promoter P trc Regulation.

2. The xanthic acid-producing strain according to claim 1, characterized in that: In the genome gapC Site-integrated from B. subtilis 168 amide phosphoribosyltransferase gene Using the promoter P trc Regulation; in the genome yjgX Integrating phosphoribosyl pyrophosphate synthase gene at the site prs D128A Using the promoter P trc Regulation; in the genome mbhA IMP dehydrogenase gene integrated at the site guaB Using the promoter P T7 Regulation; in the genome ylbE Integrating hypoxanthine phosphoribosyltransferase gene at the site hpt Using the promoter P trc Regulation; in the genome yncI Integrating guanine hypoxanthine phosphoribosyltransferase gene at the site gpt Using the promoter P trc Regulation; in the genome ilvG The artificial operon PUR is integrated in segments at the site using the promoter P. trc Regulation.

3. The xanthic acid-producing strain according to claim 1 or 2, characterized in that: The nucleotide sequence of the artificial operon PUR is shown in SEQ ID NO.

11.

4. The xanthic acid-producing strain according to claim 1, characterized in that: The surE The nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing. ushA The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing. nagD The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing. purR The nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing; guaA The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing; prs D128A The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing. guaB The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing. hpt The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing. gpt The nucleotide sequence is shown in SEQ ID NO. 10 of the sequence listing; the promoter P fliC The nucleotide sequence is shown in the sequence listing SEQ ID NO.

14.

5. The xanthic acid-producing strain according to claim 1 or 2, characterized in that: The P trc The nucleotide sequence of the promoter is shown in SEQ ID NO. 12 of the sequence listing; P T7 The nucleotide sequence of the promoter is shown in SEQ ID NO. 13 of the sequence listing.

6. The method for constructing the xanthic acid-producing strain according to any one of claims 1-5, characterized in that: The specific steps are as follows: (1) In E. coli Knockout of strain W3110 surE , ushA、nagD as well as purR Gene; (2) In the genome gapC and yjgX Overexpression at each site ,prs D128A and with P trc Promoters regulate separately; in the genome mbhA Overexpression at site guaB, and use P T7 Promoter regulation; (3) In the genome ylbE、yncI Overexpression at each site hpt, gpt and with P trc Promoter regulation; (4) Using the E. coli flagellar filament structural protein gene fliC promoter P fliC Replace the GMP synthase gene in the genome guaA The natural promoter; (5) In ilvG The artificial operon PUR is integrated in segments at the site and then used with P trc The promoter is used for regulation.

7. The use of the xanthocyanidin-producing strain according to any one of claims 1-5 in the production of xanthocyanidins.

8. The application according to claim 7, characterized in that: Xanthocyanidins are produced through fermentation.

9. The application according to claim 7 or 8, characterized in that: Xanthocyanidins were obtained by shake-flask culture, and the specific steps are as follows: (1) Activation of bacterial strain: Inoculate the bacterial solution from the preservation tube into the slant medium for activation culture; transfer the colonies from the slant medium to LB medium for further activation culture; (2) Inoculate 10%-15% into the fermentation medium, seal and shake to culture. During fermentation, maintain pH at 7.0-7.2 by adding ammonia water; add glucose solution to maintain fermentation.

10. The application according to claim 7 or 8, characterized in that: The specific steps for producing xanthic acid using a fermenter are as follows: 1) Seed activation: After the inoculum is evenly spread on the activation slant and cultured, it is transferred to a flask for further culture; 2) Seed culture: The bacterial suspension was inoculated into the seed culture medium and cultured at pH 7.0, constant temperature of 36℃, and dissolved oxygen of 30%-60%. 3) Fermentation culture: wait for the seed cell volume OD 600 to 20-25, according to the 20% inoculation amount into the fermentation medium, start fermentation, control the pH 7.0, the temperature 36℃, the dissolved oxygen in 30%-60% during the fermentation process; when the glucose in the medium is consumed, glucose solution is added to maintain the glucose concentration in the fermentation medium at 0.1-1g / L.

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