Genetically engineered bacterium for efficiently synthesizing betacyanin as well as construction method and application of genetically engineered bacterium

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium for efficiently synthesizing betacyanin as well as a construction method and application of the genetically engineered bacterium. According to the invention, a gene editing technology is used for jointly enhancing the expression levels of phosphoglucomutase pgm and UDP-glucose pyrophosphorylase galU on a BL21 (DE3) genome, the supply level of uridine diphosphate glucose which is a precursor substance for synthesizing betacyanin can be effectively improved, and a genetically engineered bacterium is constructed by combining a double-plasmid multienzyme expression system and overexpressing hpaBC, dodA, CYP76AD1 and 5GT. The engineering bacterium realizes whole-cell catalytic synthesis of betacyanin by taking tyrosine and glucose as direct substrates for the first time through collaborative optimization of genome editing and a plasmid expression system, and the product yield and the process economy are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium for efficiently synthesizing betalain, and a construction method and application thereof. BACKGROUND

[0002] Betalain is a natural water-soluble pigment, which is widely present in plants such as sugar beet and amaranth, has bright red color and good stability, and is widely used in food, cosmetics, medicine and other fields. At present, the yield of betalain obtained by the existing plant extraction method is only about 0.3%, and there are problems such as strong dependence on raw materials and complex process. In recent years, although there has been a breakthrough in microbial fermentation method (such as the yield of 1.91 g / L reported by Tianjin Industrial Biology), there are still bottlenecks such as insufficient supply of precursors, strong dependence on coenzyme and long fermentation period.

[0003] In the prior art, there is no scheme for strengthening the endogenous precursor synthesis pathway by gene editing and integrating a multi-enzyme expression system to realize the whole-cell catalytic synthesis of betalain. The existing researches are mostly focused on the optimization and expression of a single or a few key enzymes, and lack of systematic and overall modification of the entire biosynthesis pathway, which cannot fully exert the potential of microbial cell factories and realize the efficient synthesis of betalain. Therefore, it is of urgent practical needs and important scientific significance to develop a brand-new and efficient method for synthesizing betalain. SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art and provide a genetically engineered bacterium for efficiently synthesizing betalain and a construction method and application thereof, so as to solve the problems of limited raw materials, low yield of traditional plant extraction method, insufficient supply of precursors, strong dependence on coenzyme and long fermentation period of microbial fermentation method, and significantly improve the synthesis efficiency of betalain.

[0005] To achieve the above object, the technical scheme adopted by the present application comprises:

[0006] In a first aspect, the present application provides a genetically engineered bacterium for efficiently synthesizing betalain, which uses Escherichia coli as a starting strain, and adopts a constitutive strong promoter P J23119 to regulate the expression of phosphoglucomutase gene pgm and / or UDP-glucose pyrophosphorylase gene galU, and overexpresses 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC, 4,5-dioxygenase gene dodA, ring dopa synthase gene CYP76AD1 and 5-O-glucosyltransferase gene 5GT in the starting strain.

[0007] Preferably, a constitutive strong promoter P J23119 is used to regulate the expression of phosphoglucomutase gene pgm and UDP-glucose pyrophosphorylase gene galU, respectively.

[0008] The application uses gene editing technology (Crispr / Cas9) to co-enhance the expression levels of phosphoglucomutase pgm and UDP-glucose pyrophosphorylase galU on the genome of Escherichia coli, which can effectively improve the supply level of the precursor substance of betalain, uridine diphosphate glucose (UDP-glucose), and combine a double-plasmid multi-enzyme expression system to overexpress 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC, 4,5-dopa dioxygenase gene dodA, ring dopa synthetase gene CYP76AD1 and 5-O-glucosyltransferase gene 5GT, and a genetically engineered bacterium is constructed. The engineered bacterium is optimized by genome editing and plasmid expression system, which realizes the whole-cell catalytic synthesis of betalain with tyrosine and glucose as direct substrates for the first time, and significantly improves the yield of the product and the process economy.

[0009] Preferably, the constitutive strong promoter P J23119 The nucleotide sequence of the constitutive strong promoter P is shown in SEQ ID NO. 1.

[0010] Preferably, the Escherichia coli is Escherichia coli BL21 (DE3).

[0011] Preferably, the phosphoglucomutase gene pgm is an endogenous gene of BL21 (DE3), and the gene NCBI sequence number is ECK0676.

[0012] Preferably, the UDP-glucose pyrophosphorylase gene galU is an endogenous gene of BL21 (DE3), and the gene NCBI sequence number is ECK1231.

[0013] Preferably, the nucleotide sequence of the 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC is shown in SEQ ID NO. 2, the nucleotide sequence of the 4,5-dopa dioxygenase gene dodA is shown in SEQ ID NO. 3, the nucleotide sequence of the ring dopa synthetase gene CYP76AD1 is shown in SEQ ID NO. 4, and the nucleotide sequence of the 5-O-glucosyltransferase gene 5GT is shown in SEQ ID NO. 5.

[0014] Preferably, the 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC is derived from Escherichia coli, the 4,5-dopa dioxygenase gene dodA is derived from the gomphid fungus, and the ring dopa synthetase gene CYP76AD1 and the 5-O-glucosyltransferase gene 5GT are both derived from Beta vulgaris.

[0015] In a second aspect, the application provides a construction method of the genetically engineered bacterium, comprising the following steps:

[0016] S1, connecting hpaBC and dodA to plasmid I to obtain recombinant plasmid I, and then connecting CYP76AD1 and 5GT to plasmid II to obtain recombinant plasmid II;

[0017] S2, replacing the upstream promoter of the pgm and / or galU sequence in the E. coli genome with a strong constitutive promoter P J23119 to obtain engineering bacteria I;

[0018] S3, co-transferring the recombinant plasmid I and the recombinant plasmid II into the engineering bacteria I to obtain the genetically engineered bacteria.

[0019] Preferably, the plasmid I comprises pRSFDuet, and the plasmid II comprises pCDFDuet. The genotype of the recombinant plasmid I is pRSFDuet-hpaBC-dodA, and the genotype of the recombinant plasmid II is pCDFDuet-CYP76AD1-5GT. The engineering bacteria I is HG-Bet01 or HG-Bet02 involved in the specific embodiments.

[0020] Preferably, the upstream promoter of the pgm sequence in the E. coli genome is replaced with a strong constitutive promoter P J23119 The nucleotide sequences of the primer sets involved are shown in SEQ ID NO. 6-13; the upstream promoter of the galU sequence in the E. coli genome is replaced with a strong constitutive promoter P J23119 The nucleotide sequences of the primer sets involved are shown in SEQ ID NO. 6-13; the upstream promoter of the galU sequence in the E. coli genome is replaced with a strong constitutive promoter P

[0021] In a third aspect, the present application provides a whole-cell catalyst, which comprises the genetically engineered bacteria.

[0022] In a fourth aspect, the present application provides an application of the genetically engineered bacteria in the production of betalains.

[0023] Preferably, the application method comprises the following steps:

[0024] S1, performing shaker culture on the genetically engineered bacteria, then adding IPTG to induce protein expression, continuing to culture, centrifuging to collect the bacterial cells, and obtaining a whole-cell catalyst;

[0025] S2, adding the whole-cell catalyst into a PBS buffer containing tyrosine, glucose and ascorbic acid for culture.

[0026] The application provides a method for synthesizing betalain by whole cell catalysis reaction with tyrosine and glucose as substrates and ascorbic acid as a cofactor, compared with chemical synthesis, the whole cell catalytic synthesis avoids the use of toxic reagents; compared with plant extraction, the whole cell catalytic synthesis is not limited by the planting conditions of plants, and the production efficiency is higher. In addition, the raw materials of the method are easy to obtain, the reaction conditions are mild, and the product purity is high, which provides a new way for the industrialized production of betalain. And the betalain synthesized by the application can be widely used in food, cosmetics, medicine and other fields, and has broad market prospect.

[0027] Preferably, in step S2, the temperature of the culture is 25-40℃, the pH value of the culture is 6-7.5, and the bacterial amount of the whole cell catalyst is 10-50OD 600 Here, the bacterial amount is 10-50OD 600 , which means the product of OD 600 value and bacterial liquid volume, which can be understood as 10mL OD 600 =1 bacterial liquid or 5mL OD 600 =2 bacterial liquid, that is, the whole cell catalyst OD 600 is 10, and the bacterial body is collected by centrifugation and then cultured in the buffer.

[0028] More preferably, the temperature of the culture is 30℃, the pH value of the culture is 6.5, and the bacterial amount of the whole cell catalyst is 30OD 600 .

[0029] It is found through experiments that when the whole cell catalyst prepared by the application is used for the synthesis of betalain, the culture conditions involved have an important influence on the yield of betalain, and when the culture conditions are accurately controlled to the above optimal values, the yield of betalain can be significantly increased to 1216mg / L, reaching the highest level of the prior art, and having a significant technical advantage.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] (1) The application replaces the constitutive strong promoter on the E. coli genome by CRISPR / Cas9 gene editing technology, significantly enhances the expression level of phosphoglucomutase gene pgm and UDP-glucose pyrophosphorylase gene galU, and effectively improves the supply capacity of betalain synthesis key precursor substance-uridine diphosphate glucose (UDP-glucose), which lays a material foundation for efficient synthesis of betalain;

[0032] (2) The application also combines a double-plasmid multi-enzyme expression system to overexpress 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC, 4,5-dihydroxybenzoate dioxygenase gene dodA, cyclo-DOPA synthase gene CYP76AD1 and 5-O-glucosyltransferase gene 5GT, realizes efficient expression and synergistic catalysis of key enzymes in the betalain synthesis pathway, and effectively improves the overall efficiency of the synthesis reaction taking tyrosine as the substrate;

[0033] (3) The application realizes efficient synthesis of betalain by the synergistic optimization of genome editing and plasmid expression system, and the genetically engineered bacteria have the ability to efficiently synthesize betalain, which has a significant improvement in synthesis rate, product yield and stability compared with traditional synthesis methods, has strong industrial application potential, and provides a reliable strain basis for large-scale production of betalain;

[0034] (4) The application realizes the whole-cell catalytic synthesis of betalain by taking tyrosine and glucose as direct substrates for the first time, without the need for additional complex intermediate products, and the reaction conditions are mild, the yield of betalain can be significantly improved to 1216mg / L, reaching the highest level of the prior art, and the product has high purity, meeting the needs of food, cosmetics, medicine and other fields for high-quality betalain. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The figure is a betalain synthesis pathway constructed by the application;

[0036] Figure 2 The figure is a betalain standard HPLC detection result;

[0037] Figure 3 The figure is a betalain production strain HG-Bet03 catalytic liquid HPLC detection result constructed by the application. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and effect of the application more clear and explicit, the following examples are used to further describe the application. It should be understood that the specific examples described herein are only used to explain the application, and are not used to limit the application.

[0039] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0040] Escherichia coli DH5α (hereinafter referred to as DH5α) was used for vector construction, and Escherichia coli BL21 (DE3) was purchased from GenScript Biotech Corporation; plasmids pTargetF, pCas9, pRSFDuet and pCDFDuet were purchased from BioWind Corporation; high-fidelity DNA polymerase and In-Snap Assembly Master Mix were purchased from TAKARA; plasmid extraction kit, DNA purification kit and gel recovery kit were purchased from OMEGA.

[0041] LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, add water to a total volume of 1 L, sterilize at 121℃ for 20 min; if prepared as a solid medium, add 15-20 g / L agar powder.

[0042] The primer sequences involved in the following examples of the application are shown in Table 1.

[0043] Table 1

[0044]

[0045]

[0046] The strain gene information involved in the following examples of the application is shown in Table 2.

[0047] Table 2

[0048]

[0049] The sample concentration of betalain in the following examples was detected by high performance liquid chromatography as follows.

[0050] Chromatographic conditions:

[0051] Mobile phase: 0.1% formic acid aqueous solution and methanol gradient elution, gradient table 3 as follows:

[0052] Wavelength 535 nm, flow rate 1.0 ml / min, sample solution: DMSO, injection volume: 10 μL, column temperature 35℃, running time 30 min;

[0053] Chromatographic column: EF-C18M 4.6mm id x 250mm L (SN B06211801).

[0054] Table 3

[0055] Time min A (0.1% formic acid water) % B (methanol) % 0 90 10 10 70 30 20 50 50 25 90 10 30 90 10

[0056] Example 1

[0057] The present embodiment provides an engineered bacterium HG-Bet01 for synthesizing betacyanin, and a construction method thereof, which comprises the following steps.

[0058] 1.1 Construction of pTarget pgm plasmid

[0059] The pTarget pgm plasmid was constructed by using the plasmid pTargetF as a template, performing PCR amplification by using the primers pT pgm-F and pT pgm-R, and performing Dpn I enzyme digestion on the PCR product, and then transforming the product into DH5α.

[0060] 1.2 Construction of PJ23119-pgm targeting fragment

[0061] (1) The PJ23119-pgm-Up fragment was obtained by performing PCR amplification by using the BL21(DE3) as a template and the primers pgm U800-F and pgm U800-R, wherein the primer pgm U800-R contains the promoter PJ23119 sequence as shown in SEQ ID NO. 1;

[0062] (2) The PJ23119-pgm-Down fragment was obtained by performing PCR amplification by using the BL21(DE3) as a template and the primers pgm D800-F and pgm D800-R, wherein the primer pgm D800-F contains the promoter PJ23119 sequence;

[0063] (3) The PJ23119-pgm targeting fragment was obtained by performing PCR amplification by using the primers pgm U500-F and pgm D500-R and by using the PJ23119-pgm-Up fragment and the PJ23119-pgm-Down fragment as templates;

[0064] 1.3 Construction of HG-Bet01 strain

[0065] (1) The BL21(DE3) / pCas9 strain was obtained by transforming the pCas9 plasmid into the BL21(DE3) by using the calcium chloride transformation method, wherein the pCas9 plasmid was purchased from BioWind Company;

[0066] (2) The pTarget-pgm plasmid obtained in step 1.1 and the PJ23119-pgm targeting fragment obtained in step 1.2 were co-transformed into the BL21(DE3) / pCas9 strain by using the electroporation method, and the target strain BL21(DE3) / PJ23119-pgm, named HG-Bet01, was obtained by colony PCR screening.

[0067] Example 2

[0068] The present embodiment provides an engineered bacterium HG-Bet02 for synthesizing betacyanin, and a construction method thereof, which comprises the following steps:

[0069] 2.1 Construction of pTarget galU plasmid

[0070] The pTarget galU plasmid is obtained by using the pTargetF plasmid as a template, performing PCR amplification by using the pT galU-F and pT galU-R primers, performing Dpn I enzyme digestion on the PCR product, and then transforming DH5α.

[0071] 2.2 Construction of PJ23119-galU targeting fragment

[0072] (1) The PJ23119-galU-Up fragment is obtained by using BL21(DE3) as a template and performing PCR amplification by using the galU U800-F and galU U800-R primers, wherein the primer galU U800-R contains the promoter PJ23119 sequence;

[0073] (2) The PJ23119-galU-Down fragment is obtained by using BL21(DE3) as a template and performing PCR amplification by using the galU D800-F and galU D800-R primers, wherein the primer galU D800-F contains the promoter PJ23119 sequence;

[0074] (3) The PJ23119-galU targeting fragment is obtained by using the PJ23119-galU-Up fragment and the PJ23119-galU-Down fragment as templates and performing PCR amplification by using the galU U500-F and galU D500-R primers.

[0075] 2.3 Construction of HG-Bet02 strain

[0076] The pTarget-galU plasmid obtained in step 2.1 and the PJ23119-galU targeting fragment obtained in step 2.2 are co-transformed into the HG-Bet01 strain obtained in Example 1 by using an electroporation method, and the target strain HG-Bet01 PJ23119-galU is obtained by colony PCR screening, which is named as HG-Bet02.

[0077] Example 3

[0078] The present embodiment provides a construction method of a recombinant plasmid pRSFDuet-hpaBC-dodA, which comprises the following steps:

[0079] 3.1 Construction of pRSFDuet-hpaBC recombinant plasmid

[0080] (1) Using the genome of Escherichia coli BL21(DE3) as a template, PCR amplification was performed using pRSFDuet-hpaBC-F and pRSFDuet-hpaBC-R as primers to obtain the hpaBC fragment encoding the 4-hydroxyphenylacetate 3-hydroxylase complex (nucleotide sequence as shown in SEQ ID NO: 2).

[0081] (2) Using the commercial plasmid pRSFDuet-1 purchased from Biowind as a template, PCR amplification was performed using primers pRSF-I-F1 and pRSF-I-R1, and the product was purified to obtain the linearized vector of pRSFDuet-1.

[0082] (3) The pRSFDuet-1 linearized vector and hpaBC gene fragment were ligated using the seamless cloning ligation kit of Takara Bio Inc. The ligation product was transformed into Escherichia coli DH5α by chemical transformation. After recovery and culture, it was plated on LB solid medium plates containing 50 μg / mL kanamycin resistance and incubated at 37°C for about 16 h.

[0083] (4) Use primers pRSF-YZ-F1 and pRSF-YZ-R1 to perform colony PCR verification. Culture the strains that are verified correctly by PCR, extract the recombinant plasmid, and send it to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid that is correctly sequenced is pRSFDuet-hpaBC.

[0084] 3.2 Construction of pRSFDuet-hpaBC-dodA recombinant plasmid

[0085] (1) Using pRSFDuet-hpaBC plasmid as a template, PCR amplification was performed using pRSF-I-F2 and pRSF-I-R2, and the product was purified to obtain the linearized vector pRSFDuet-hpaBC.

[0086] (2) 4,5-DOPA dioxygenase (dodA) is derived from Amanita thiersii Skay4041. The nucleotide sequence dodA was synthesized by Qingke Biotechnology after codon optimization. Homologous arms of the vector were added to both ends of the gene during gene synthesis. The nucleotide sequence is shown in SEQ ID NO: 3.

[0087] (3) The pRSFDuet-hpaBC linearized vector and dodA fragment were ligated using the Takara Bio Seamless Cloning Ligation Kit. The ligation product was then transformed into Escherichia coli DH5α using chemical transformation. After resuscitation and culture, the product was plated onto LB solid medium plates containing 50 μg / mL kanamycin resistance and incubated at 37°C for about 16 h.

[0088] (4) Using primers pRSF-YZ-F2 and pRSF-YZ-R2, colony PCR verification was performed, and the strains correctly verified by PCR were cultured, the recombinant plasmid was extracted, and was sent to Qianke Biotechnology Company for sequencing. The correct sequencing was pRSFDuet-hpaBC-dodA plasmid.

[0089] Example 4

[0090] The present embodiment provides a construction method of a recombinant plasmid pCDFDuet-CYP76AD1-5GT, which comprises the following steps:

[0091] 4.1 Construction of recombinant plasmid pCDFDuet-CYP76AD1

[0092] (1) The cyclo-Dopa synthase (CYP76AD1) is derived from Beta vulgaris, and is synthesized by Qianke Biotechnology Company after codon optimization. (The homologous arm of the vector is added at both ends of the gene during gene synthesis), and the nucleotide sequence is shown in SEQ ID NO. 4;

[0093] (2) Using the commercial plasmid pCDFDuet-1 purchased from Haibo Biotechnology as a template, primers pCDF-I-F1 and pCDF-I-R1 were used for PCR amplification, and the product was purified to obtain a pCDFDuet-1 linearized vector;

[0094] (3) The pCDFDuet-1 linearized vector and CYP76AD1 fragment were connected using a seamless cloning kit, and the ligation product was transformed into E. coli DH5α by chemical transformation method. After recovery and culture, it was coated on LB solid medium containing 50 μg / mL of streptomycin resistance, and cultured in a 37°C incubator for about 16 hours;

[0095] (4) Using primers pCDF-YZ-F1 and pCDF-YZ-R1, colony PCR verification was performed, and the strains correctly verified by PCR were cultured, the recombinant plasmid was extracted, and was sent to Qianke Biotechnology Company for sequencing. The correct sequencing was pCDF-CYP76AD1 plasmid.

[0096] 4.2 Construction of recombinant plasmid pCDFDuet-CYP76AD1-5GT

[0097] (1) Using pCDF-CYP76AD1 plasmid as a template, pCDF-I-F2 and pCDF-I-R2 were used for PCR amplification, and the product was purified to obtain a pCDF-CYP76AD1 linearized vector;

[0098] (2) 5-O-glucosyltransferase (5GT) is derived from Beta vulgaris, and is obtained by synthesis after codon optimization by Genescript Biotech Co., Ltd. The homologous arms of the vector are added at both ends of the gene during gene synthesis. The nucleotide sequence is shown as SEQ ID NO. 5;

[0099] (3) The pCDF-CYP76AD1 linearized vector and the 5GT gene fragment are connected using a seamless cloning kit. The ligation product is transformed into E. coli DH5α by chemical transformation method. After recovery and culture, it is plated on LB solid medium containing 50 ug / mL of streptomycin resistance and cultured in a 37℃ incubator for about 16 hours.

[0100] (4) Colony PCR verification is performed using primers pCDF-YZ-F2 and pCDF-YZ-R2. The correct strain is cultured, the recombinant plasmid is extracted, and sent to Genescript Biotech Co., Ltd. for sequencing. The correct sequencing is the recombinant plasmid pCDFDuet-CYP76AD1-5GT.

[0101] Example 5

[0102] The present embodiment provides an engineered bacterium HG-Bet03 for synthesizing betalains, and the construction method thereof comprises the following steps:

[0103] The pRSFDuet-hpaBC-dodA plasmid constructed in Example 3 and the pCDFDuet-CYP76AD1-5GT plasmid constructed in Example 4 are co-transformed into E. coli HG-Bet02 by calcium chloride transformation method to obtain the recombinant strain HG-Bet02 / pRSFDuet-hpaBC-dodA+pCDFDuet-CYP76AD1-5GT, which is named HG-Bet03.

[0104] Example 6

[0105] The present embodiment provides an engineered bacterium HG-Bet04 for synthesizing betalains, and the construction method thereof comprises the following steps:

[0106] The pRSFDuet-hpaBC-dodA plasmid constructed in Example 3 and the pCDFDuet-CYP76AD1-5GT plasmid constructed in Example 4 are co-transformed into E. coli BL21(DE3) by calcium chloride transformation method to obtain the recombinant strain BL21(DE3) / pRSFDuet-hpaBC-dodA+pCDFDuet-CYP76AD1-5GT, which is named HG-Bet04.

[0107] Example 7

[0108] The present embodiment provides an engineered bacterium HG-Bet05 for synthesizing betalain, and a construction method thereof, which comprises the following steps.

[0109] The pRSFDuet-hpaBC-dodA plasmid constructed in Example 3 and the pCDFDuet-CYP76AD1-5GT plasmid constructed in Example 4 are co-transformed into E. coli HG-Bet01 by using calcium chloride transformation method to obtain a recombinant strain HG-Bet01 / pRSFDuet-hpaBC-dodA+pCDFDuet-CYP76AD1-5GT, which is named HG-Bet05.

[0110] Example 8

[0111] The present embodiment provides a method for producing betalain, which comprises the following steps.

[0112] (1) The engineered strains HG-Bet02, HG-Bet03, HG-Bet04 and HG-Bet05 constructed in Examples 2, 5-7, respectively, and BL21(DE3) are activated, and then inoculated into 50 mL of LB medium at an inoculation amount of 1%, and cultured at a shaking speed of 220 rpm and a temperature of 30°C until the OD reaches 0.6-0.8, and then induced for protein expression by adding IPTG at a final concentration of 0.1 mM, and then cultured for another 12 h, and then centrifuged (at 4°C, 4000 rpm, for 10 min) to collect the bacterial cells, which are full-cell catalysts;

[0113] (2) Full-cell catalysis: 10 mL of PBS buffer (pH = 7.0) containing 2 g / L tyrosine, 2 g / L glucose and 1 g / L ascorbic acid is sequentially added into a 100 mL triangular flask, and then 10 OD of the full-cell catalyst is added, and then the mixture is cultured at 37°C and a shaking speed of 220 rpm for 12 h, and then sampled for HPLC detection of the concentration of betalain in the catalytic liquid. 600 The full-cell catalyst is cultured at 37°C and a shaking speed of 220 rpm for 12 h, and then sampled for HPLC detection of the concentration of betalain in the catalytic liquid.

[0114] The PBS buffer formula is as follows: 8.0 g / L Nacl, 0.2 g / L KCL, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, and pH = 7.0 adjusted by concentrated HCl.

[0115] As shown in Table 4, the starting strain BL21(DE3) cannot synthesize betalain, and the yield of HG-Bet03 is the highest, reaching 264 mg / L after 12 h, and the betalain synthesis pathway is as shown in Figure 1 The HPLC detection result of the betalain standard is as shown in Figure 2 The HPLC detection result of the catalytic liquid of the betalain-producing strain HG-Bet03 is as shown inFigure 3 Time min A (0.1% formic acid water) % B (methanol) % Figure 1 Figure 2 Figure 3 Time min A (0 As shown, 8.8 min is the betalain absorption peak, which is consistent with the betalain standard. HPLC results confirmed that the HG-Bet03 strain can oxidize tyrosine to levodopa by hpaBC, and levodopa is catalyzed by dodA to generate betalamic acid, and CYP76AD1 and 5GT sequentially complete cyclization and glycosylation, and finally generate betalains. By comparison, the highest yield of betalain-producing strain HG-Bet03 (betalain yield 264 mg / L) was obtained, which was 5 times higher than the control strain HG-Bet04 (52 mg / L). The gene editing of HG-Bet03 significantly improved the precursor supply efficiency, and verified the key role of endogenous strengthening strategy.

[0116] Table 4

[0117]

[0118] Example 9

[0119] This example provides a method for generating betalains, and explores the effect of different reaction temperatures on whole cell catalysis. The specific method is as follows:

[0120] (1) Activation of the engineered strain HG-Bet03 constructed in Example 5, inoculated into 50 mL LB medium at 1% inoculation amount, the shaking bed temperature was 30°C, the rotation speed was 220 rpm, and the OD was cultured to 0.6-0.8. Add 0.1 mM IPTG to induce protein expression, continue to culture for 12 h, centrifuge (4°C, 4000 rpm, 10 min) to collect the bacterial cells, which are whole cell catalysts;

[0121] (2) Whole cell catalysis: 10 mL of PBS buffer (pH = 7.0) containing 2 g / L tyrosine, 2 g / L glucose, and 1 g / L ascorbic acid was added to a 100 mL flask, and 10 OD 600 Whole cell catalyst, under the condition of 20-40°C, 220 rpm shaking bed culture for 12 h, sample HPLC to detect the concentration of betalains in the catalytic liquid.

[0122] Among them, the PBS buffer formula mentioned is: 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, adjusted to pH = 7.0 with concentrated HCl.

[0123] Among them, the reaction temperature mentioned is 20-40°C, specifically 20°C, 25°C, 30°C, 37°C and 40°C.

[0124] The results are shown in Table 5. Under the same conditions, 30°C is the optimum temperature, at which the betalain yield is the highest, reaching 344 mg / L.

[0125] Table 5

[0126]

[0127] Example 10

[0128] This example provides a method for generating betalain, and explores the influence of different reaction pH on the catalytic effect of whole cell, and the specific method is as follows:

[0129] (1) Activate the engineered strain HG-Bet03 constructed in Example 5 with a 1% inoculation amount into 50 mL of LB medium, with a shaking bed temperature of 30°C and a rotation speed of 220 rpm. Culture to OD 0.6-0.8, add 0.1 mM IPTG to induce protein expression, continue to culture for 12 h, centrifuge (4°C, 4000 rpm, 10 min) to collect the bacterial cells, which are whole cell catalysts;

[0130] (2) Whole cell catalysis: add 10 mL of PBS buffer (pH = 6.0-8.0) containing 2 g / L tyrosine, 2 g / L glucose, and 1 g / L ascorbic acid into a 100 mL flask, and then add 10 OD 600 Whole cell catalysts, under the condition of 30°C and 220 rpm shaking bed culture for 12 h, sample HPLC to detect the concentration of betalain in the catalytic liquid.

[0131] Among them, the PBS buffer formula mentioned is: 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, adjust to pH = 6.0-8.0 with concentrated HCl, and the specific values are 6.0, 6.5, 7.0, 7.5, and 8.0, respectively.

[0132] The results are shown in Table 6. Under the same conditions, pH = 6.5 is the optimum pH, at which the betalain yield is the highest, reaching 406 mg / L.

[0133] Table 6

[0134]

[0135]

[0136] Example 11

[0137] This example provides a method for generating betalain, and explores the influence of different whole cell catalyst addition amounts on the catalytic effect of whole cell, and the specific method is as follows:

[0138] (1) The engineered strain HG-Bet03 constructed in Example 5 was activated and inoculated into 50 mL LB medium at an inoculation amount of 1%, and cultured at 30°C and 220 rpm until the OD reached 0.6-0.8, then 0.1 mM IPTG was added to induce protein expression, and the culture was continued for 12 h. The bacterial cells were collected by centrifugation (4°C, 4000 rpm, 10 min) to obtain the whole-cell catalyst;

[0139] (2) Whole-cell catalysis: 10 mL of PBS buffer (pH 6.5) containing 2 g / L tyrosine, 2 g / L glucose, and 1 g / L ascorbic acid was added to a 100 mL flask, followed by the addition of 5-50 OD 600 The whole-cell catalyst was cultured at 30°C and 220 rpm for 12 h, and the concentration of betalain in the catalytic solution was detected by HPLC.

[0140] The PBS buffer mentioned above has the following formulation: 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, and pH 6.5 adjusted with concentrated HCl.

[0141] The bacterial amount mentioned above is 5-50 OD 600 , specifically 5 OD 600 , 10 OD 600 , 20 OD 600 , 30 OD 600 , 40 OD 600 , or 50 OD 600 .

[0142] The results are shown in Table 7. Under the same conditions, the optimal amount of whole-cell catalyst is 30 OD 600 , at which the yield of betalain reaches a maximum of 1216 mg / L. When the bacterial amount is 30 OD 600 , the catalytic efficiency is at the best balance, and excessive bacterial cells lead to substrate competition inhibition.

[0143] Table 7

[0144]

[0145] In conclusion, the pgm and galU genes of the host bacteria are strengthened by the CRISPR / Cas9 technology, and four key enzymes are overexpressed by a double-plasmid system, so that one-step efficient conversion is realized by taking tyrosine and glucose as substrates. Under the optimized process, the betalain yield reaches 1216 mg / L, and the molar conversion rate reaches 20.6% at 30 DEG C and pH 6.5 within 12 hours, which reaches the highest level of the prior art. The method has the advantages of cheap and easily available raw materials, mild reaction, short reaction period and the like, and is suitable for industrial production.

[0146] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A genetically engineered bacterium that efficiently synthesizes betaine, characterized in that, Using *Escherichia coli* as the starting strain, and employing the constitutive strong promoter P... J23119 The expression of the phosphogluconomutase gene pgm and / or the UDP-glucose pyrophosphorylase gene galU was regulated, and the 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC, the 4,5-DOPA dioxygenase gene dodA, the cyclic dopa synthase gene CYP76AD1, and the 5-O-glucosyltransferase gene 5GT were overexpressed in the starting strain.

2. The genetically engineered bacteria as described in claim 1, characterized in that, The constitutive strong promoter P J23119 The nucleotide sequence is shown in SEQ ID NO.

1.

3. The genetically engineered bacteria as described in claim 1, characterized in that, The nucleotide sequence of the 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC is shown in SEQ ID NO.2, the nucleotide sequence of the 4,5-DOPA dioxygenase gene dodA is shown in SEQ ID NO.3, the nucleotide sequence of the cyclic dopa synthase gene CYP76AD1 is shown in SEQ ID NO.4, and the nucleotide sequence of the 5-O-glucosyltransferase gene 5GT is shown in SEQ ID NO.

5.

4. The genetically engineered bacteria as described in claim 1, characterized in that, The 4-hydroxyphenylacetate 3-hydroxylase complex gene hpaBC is derived from Escherichia coli, the 4,5-DOPA dioxygenase gene dodA is derived from Amanita muscaria, and the cyclic dopa synthase gene CYP76AD1 and the 5-O-glucosyltransferase gene 5GT are both derived from sugar beets.

5. The method for constructing genetically engineered bacteria according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Link hpaBC and dodA to plasmid I to obtain recombinant plasmid I. Then link CYP76AD1 and 5GT to plasmid II to obtain recombinant plasmid II. S2. Replace the upstream promoter of the pgm and / or galU sequences in the *E. coli* genome with a constitutive strong promoter, P. J23119 Engineered bacteria I were obtained; S3. Recombinant plasmid I and recombinant plasmid II are jointly transferred into engineered bacteria I to obtain the genetically engineered bacteria.

6. The construction method as described in claim 5, characterized in that, Plasmid I includes pRSFDuet, and plasmid II includes pCDFDuet.

7. A whole-cell catalyst, characterized in that, The whole-cell catalyst comprises the genetically engineered bacteria according to any one of claims 1-4.

8. The use of the genetically engineered bacteria as described in any one of claims 1-4 in the production of betaine.

9. The application as described in claim 8, characterized in that, The application method includes the following steps: S1. The genetically engineered bacteria were cultured in a shaker, then IPTG was added to induce protein expression. After further culture, the bacterial cells were collected by centrifugation to obtain whole-cell catalysts. S2. Simply add the whole-cell catalyst to a PBS buffer containing tyrosine, glucose, and ascorbic acid and culture.

10. The application as described in claim 9, characterized in that, In step S2, the culture temperature is 25-40℃, the culture pH is 6-7.5, and the whole-cell catalyst bacterial count is 10-50 OD. 600 .

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