Genetically engineered bacterium for synthesizing hydroxyl salidroside as well as preparation method and application of genetically engineered bacterium

Abstract

The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to a genetically engineered bacterium for synthesizing hydroxyl salidroside as well as a preparation method and application of the genetically engineered bacterium. The genetically engineered bacterium is obtained by knocking out a PPC enzyme, a TyrR enzyme, a TyrE enzyme, a PheA enzyme and a feaB enzyme on an escherichia coli genome, overexpressing an ACS enzyme, an aceA enzyme, a pck enzyme, a ppsA enzyme, an fbp enzyme, a pgm enzyme, an HpaC enzyme, a galU enzyme and a mutated TyrAfbr enzyme and an AroGfbr enzyme, and introducing an ADH6 enzyme, an ARO10 enzyme, an RrUGT33 enzyme and an HpaB enzyme. The genetically engineered bacterium can efficiently synthesize the hydroxyl salidroside by taking acetate as a unique carbon source, the yield of a 5L fermentation tank reaches 6.3 g / L, the conversion rate is 0.45 g / g, the production cost is remarkably reduced, the substrate toxicity is avoided, and a new strategy is provided for industrial production of the hydroxyl salidroside and high-value utilization of acetate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and microbial technology, and particularly relates to a genetically engineered bacterium for synthesizing hydroxysalidroside as well as a preparation method and application thereof. BACKGROUND

[0002] Rhodiola is a precious traditional Chinese medicine and is included in the 2020 edition of Chinese Pharmacopoeia. Rhodioloside, as an effective active ingredient of Rhodiola, has multiple physiological activities such as protecting cardiovascular and cerebrovascular, anti-fatigue, anti-depression, anti-aging, anti-hypoxia, anti-radiation, anti-tumor, immune regulation, and whitening and freckle-removing. It is often used in the treatment of complications of diabetes, hypoxic diseases, high altitude reaction, cardiovascular diseases, nervous system diseases, and reproductive diseases. Hydroxysalidroside is a downstream derivative of rhodioloside, and its chemical formula is C 14 H 20 O8. Hydroxysalidroside has good solubility and high bioavailability, and has the effects of anti-fatigue, anti-aging, anti-tumor, and anti-radiation, thus having a broad application prospect in the fields of food and medicine.

[0003] At present, the synthesis of hydroxysalidroside mainly includes chemical synthesis and biological synthesis. The chemical synthesis routes reported in the existing literature have certain defects, for example: ① long steps: multiple protection and deprotection reactions are required; ② low yield: generally less than 30%; ③ complex by-products: isomers are difficult to separate; ④ involves toxic reagents: such as halogenated hydrocarbons, strong acid catalysts, etc., which can easily cause environmental pollution and other problems. The biological synthesis method of hydroxysalidroside has been reported in many existing technologies, such as CN118497303A, CN117965500A, CN116355776A, CN114317480A, CN114350628A, CN112501194A, etc. However, these existing technologies mostly use hydroxytyrosol, tyrosine, etc. as the substrate to synthesize hydroxysalidroside under the catalysis of enzymes. Hydroxytyrosol is expensive, and the cost of commercially available hydroxytyrosol with a purity of ≥98% is about 3500 yuan / kg; and high concentrations are toxic to host cells (such as Escherichia coli and Saccharomyces cerevisiae), which leads to inhibition of bacterial growth and limitation of metabolic flux during fermentation. Tyrosine is relatively less expensive, about 63 yuan / kg. In addition, the insufficient intracellular supply of UDP-glucose also restricts the glycosylation efficiency, which is not conducive to large-scale production.

[0004] Therefore, it is necessary to develop a low-cost, environmentally friendly, non-toxic, and efficient method for large-scale industrial production of hydroxysalidroside. SUMMARY

[0005] In view of this, in order to solve the problems of long steps, low yield and environmental pollution of the existing chemical synthesis method, and the problems of dependence on expensive substrate hydroxytyrosol and insufficient supply of UDP-glucose of the biological synthesis method, the present application provides a genetically engineered bacterium for synthesizing hydroxy salidiloside, and a preparation method and application thereof. The genetically engineered bacterium can efficiently produce hydroxy salidiloside by utilizing acetate, and has the characteristics of low cost, environmental protection and non-toxicity.

[0006] One of the purposes of the present application is to provide a genetically engineered bacterium for synthesizing hydroxy salidiloside, which provides support for subsequent production of hydroxy salidiloside by utilizing acetate as a substrate.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The genetically engineered bacterium for synthesizing hydroxy salidiloside is obtained by knocking out PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme on the genome of Escherichia coli, overexpressing ACS enzyme, aceA enzyme, pck enzyme, ppsA enzyme, fbp enzyme, pgm enzyme, HpaC enzyme, galU enzyme and mutated TyrA fbr enzyme and AroG fbr enzyme, and introducing ADH6 enzyme, ARO10 enzyme, RrUGT33 enzyme and HpaB enzyme.

[0009] As a preference, the construction of the TyrA fbr enzyme mutant is to mutate the methionine at the 53rd position to isoleucine and the alanine at the 354th position to valine; the construction of the AroG fbr enzyme mutant is to mutate the aspartic acid at the 146th position to asparagine.

[0010] As preferred, the nucleotide sequence of the PPC enzyme is as shown in SEQ ID NO: 37.

[0011] As preferred, the nucleotide sequence of the PPC enzyme is as shown in SEQ ID NO: 37.

[0012] As preferred, the amino acid sequence of the TyrA fbr enzyme is as shown in SEQ ID NO: 13 and the nucleotide sequence is as shown in SEQ ID NO: 15; the amino acid sequence of the AroG fbr enzyme is as shown in SEQ ID NO: 14 and the nucleotide sequence is as shown in SEQ ID NO: 16.

[0013] As preferred, the ADH6 enzyme is a codon-optimized ADH6 enzyme from Saccharomyces cerevisiae S288C, the amino acid sequence of which is as shown in SEQ ID NO: 30 and the nucleotide sequence of which is as shown in SEQ ID NO: 34.

[0014] As preferred, the ARO10 enzyme is a codon-optimized ARO10 enzyme from Saccharomyces cerevisiae S288C, the amino acid sequence of which is as shown in SEQ ID NO: 29 and the nucleotide sequence of which is as shown in SEQ ID NO: 33.

[0015] As preferred, the RrUGT33 enzyme is a codon-optimized RrUGT33 enzyme from Rhodiola rosea, the amino acid sequence of which is as shown in SEQ ID NO: 32 and the nucleotide sequence of which is as shown in SEQ ID NO: 36.

[0016] As preferred, the HpaB enzyme is a codon-optimized HpaB enzyme from Pseudomonas aeruginosa, the amino acid sequence of which is shown as SEQ ID NO: 31, and the nucleotide sequence of which is shown as SEQ ID NO: 35.

[0017] As preferred, the E. coli is E. coli BL21 strain.

[0018] As preferred, the genetically engineered bacteria is E. coli strain QJS-2538.

[0019] The second object of the present application is to provide a preparation method of the aforementioned genetically engineered bacteria.

[0020] To achieve the above-mentioned objects, the present application adopts the following technical solutions.

[0021] The preparation method of the genetically engineered bacteria comprises the following steps:

[0022] S1: constructing a gRNA expression vector targeting PPC enzyme, TyrR enzyme, TrpE enzyme, PheA enzyme and feaB enzyme;

[0023] S2: constructing TyrA fbr enzyme mutant, AroG fbr enzyme mutant, ACS enzyme, aceA enzyme, pck enzyme and ppsA enzyme into the vector to obtain a first recombinant expression vector;

[0024] S3: constructing fbp enzyme, pgm enzyme, galU enzyme, ARO10 enzyme, ADH6 enzyme, HpaB enzyme, HpaC enzyme and RrUGT33 enzyme into the vector to obtain a second recombinant expression vector;

[0025] S4: introducing the gRNA expression vector obtained in S1 into E. coli competent cells to obtain E. coli in which PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme are knocked out;

[0026] S5: transforming the first recombinant expression vector obtained in S2 and the second recombinant expression vector obtained in S3 into the E. coli in which PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme are knocked out obtained in S4 to obtain genetically engineered bacteria.

[0027] As preferred, in S1, the pTarget plasmid is used as a template.

[0028] As preferred, in S1, the primers with the nucleotide sequences shown as SEQ ID NO: 1-12 are used for amplification; and in S2, the primers with the nucleotide sequences shown as SEQ ID NO: 17-28 are used for amplification.

[0029] As a preferred solution, S1 specifically comprises: amplifying with pTarget plasmid as a template, and pTa-F1-PPC-sgRNA and pTarget-R1, pTa-R1-PPC-sgRNA and pTarget-F1, pTa-F1-TyrR-sgRNA and pTarget-R1, pTa-R1-TyrR-sgRNA and pTarget-F1, pTa-F1-TrpE-sgRNA and pTarget-R1, pTa-R1-TrpE-sgRNA and pTarget-F1, pTa-F1-PheA-sgRNA and pTarget-R1, pTa-R1-PheA-sgRNA and pTarget-F1, pTa-F1-feaB-sgRNA and pTarget-R1, pTa-R1-feaB-sgRNA and pTarget-F1 primers respectively to obtain an amplified fragment; Gibson assembly of the obtained PPC-P1 and PPC-P2, TyrR-P1 and TyrR-P2, TyrE-P1 and TyrE-P2, PheA-P1 and PheA-P2, and feaB-P1 and feaB-P2 fragments respectively to obtain gRNA expression vectors targeting PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme.

[0030] As a preferred solution, the sequences of the primers pTa-F1-PPC-sgRNA, pTa-R1-PPC-sgRNA, pTa-F1-TyrR-sgRNA, pTa-R1-TyrR-sgRNA, pTa-F1-TrpE-sgRNA, pTa-R1-TrpE-sgRNA, pTa-F1-PheA-sgRNA, pTa-R1-PheA-sgRNA, pTa-F1-feaB-sgRNA, pTa-R1-feaB-sgRNA, pTarget-F1 and pTarget-R1 are shown in SEQ ID NOs: 1-12, respectively.

[0031] As a preferred solution, the vector in S2 is pRSFDuet-1 vector; and the vector in S3 is pETDuet-1 vector.

[0032] As a preferred solution, S2 comprises the following steps:

[0033] 1) synthesizing TyrA fbr enzyme mutant and AroG fbr enzyme mutant into the pRSFDuet-1 vector between the BamHI endonuclease and EcoRI endonuclease of the vector, and connecting the two genes through an SD sequence to obtain pRSFDuet-1-TyrA fbr -AroGfbr expression vector;

[0034] 2) Using the S288C genome of Saccharomyces cerevisiae as a template, respectively using primers acs-F1 / acs-R1, aceA-F1 / aceA-R1, pck-F1 / pck-R1 and ppsA-F1 / ppsA-R1 to amplify ACS fragment, aceA fragment, pck fragment and ppsA fragment; then using primers aroG-R1 / pRSFDuet-1-F1 to amplify the vector pRSFDuet-1-TyrA fbr -AroG fbr linearization; then linearizing the ACS fragment and the pRSFDuet-1-TyrA fbr -AroG fbr linearization fragment Gibson assembly, to obtain the pRSFDuet-1-TyrA fbr -AroG fbr -acs vector;

[0035] 3) Using the aceA fragment, the pck fragment and the ppsA fragment as templates, using primers aceA-F1 / ppsA-R1 to perform overlap PCR, to obtain the aceA-pck-ppsA fragment; then using primers pRSFDuet-1-R2 / pRSFDuet-1-F2 to amplify the vector pRSFDuet-1-TyrA fbr -AroG fbr -acs vector linearization; then linearizing the aceA-pck-ppsA fragment and the pRSFDuet-1-TyrA fbr -AroG fbr -acs vector linearization fragment Gibson assembly, to obtain the first recombinant expression vector.

[0036] The first recombinant expression vector is pRSFDuet-1-TyrA fbr -AroG fbr -acs-aceA-pck-ppsA.

[0037] As preferred, in step 1), the SD sequence is as shown in SEQ ID NO: 38.

[0038] As preferred, in S2, the sequences of the primers acs-F1, acs-R1, aceA-F1, aceA-R1, pck-F1, pck-R1, ppsA-F1, ppsA-R1, aroG-R1, pRSFDuet-1-F1, pRSFDuet-1-F2 and pRSFDuet-1-R2 are as shown in SEQ ID NOs: 17-28, respectively.

[0039] As a preferred solution, S3 comprises: sequentially inserting the fbp enzyme, pgm enzyme, galU enzyme, ARO10 enzyme and ADH6 enzyme between the BamHI endonuclease and EcoRI endonuclease of the pETDuet-1 vector, sequentially inserting the HpaB enzyme, HpaC enzyme and RrUGT33 enzyme between the Fsel endonuclease and Xhol endonuclease of the pETDuet-1 vector, and connecting the gene fragments through an SD sequence to obtain a second recombinant expression vector.

[0040] The second recombinant expression vector is pETDuet-1-fbp-pgm-galU-ARO10-ADH6-HpaB-HpaC-RrUGT33.

[0041] As a preferred solution, the SD sequence in S3 is as shown in SEQ ID NO: 38.

[0042] As a preferred solution, S4 is in the construction of the E. coli BL21 with inactivated PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme, in the presence of a gRNA expression vector targeting the PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme, the Cas9 system is activated after the gRNA expression vector is transformed into the E. coli competent cells, the site-directed editing of the E. coli genome is realized, and the E. coli with inactivated PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme is obtained.

[0043] As a preferred solution, the Cas9 system is a gene editing pCas9 expression plasmid.

[0044] As a preferred solution, the pCas9 plasmid is first transformed into the E. coli before the gRNA is introduced.

[0045] A third object of the present application is to provide an application of the aforementioned genetically engineered bacteria in biosynthesis of hydroxyl salidroside.

[0046] To achieve the above object, the present application adopts the following technical solutions:

[0047] The application of the aforementioned genetically engineered bacteria in biosynthesis of hydroxyl salidroside uses acetate as a substrate.

[0048] A fourth object of the present application is to provide a method for synthesizing hydroxyl salidroside by using the aforementioned genetically engineered bacteria.

[0049] To achieve the above object, the present application adopts the following technical solutions:

[0050] The method for synthesizing hydroxyl salidroside by using the aforementioned genetically engineered bacteria uses the aforementioned genetically engineered bacteria and / or the engineered bacteria constructed by using the aforementioned preparation method as fermentation bacteria, uses acetate as a substrate, and produces hydroxyl salidroside.

[0051] In the prior art, there is no relevant patent or literature report on the synthesis of hydroxyl salidroside using acetic acid as a carbon source. In view of this technical gap, the present application realizes a technological breakthrough through multi-dimensional metabolic engineering strategies: first, the competitive metabolic branch is blocked and the metabolic blockage effect is removed through targeted gene knockout technology, then the coupling pathway of acetate metabolism and target product synthesis is constructed by introducing exogenous functional genes, and the key node genes are subjected to site-directed mutation to strengthen the metabolic flux. This system optimization scheme effectively solves the two core problems faced by microorganisms in synthesizing hydroxyl salidroside using acetic acid, namely, imbalance of intracellular metabolic flux distribution and low coupling efficiency of regulation network, and provides an innovative solution for efficient utilization of unconventional carbon sources.

[0052] As preferred, the fermentation culture conditions include: the acetate concentration is 3-8 g / L; the fermentation temperature is 30-37℃; the pH is 5.5-7; the fermentation time is 100-115 h; the aeration rate is 2.0-3.0vvm; the stirring speed is 200-900 rpm; and the dissolved oxygen concentration is 30%-50%.

[0053] As a preferred scheme, the method comprises the following steps:

[0054] (1) fermenting and culturing with the aforementioned genetically engineered bacteria, under the conditions of a fermentation temperature of 30-37℃ and a stirring speed of 170-260 rpm for 10-12 h to obtain a seed liquid;

[0055] (2) performing fermenter fermentation on the seed liquid obtained in step (1), under the conditions of a fermentation temperature of 30-37℃, an aeration rate of 2.0-3.0vvm, and a stirring speed of 200-900 rpm, with a dissolved oxygen concentration of 30%-50% during the fermentation process, and by feeding acetate to control the acetate concentration at 3-6 g / L in the early stage, until OD 600 reaches 15-20, then adding IPTG to a final concentration of 0.15-0.3 mM for induction, and feeding acetate to control the acetate concentration at 5-8 g / L, to obtain hydroxyl salidroside.

[0056] As preferred, in step (1), the fermentation temperature is 35-37℃.

[0057] As preferred, in step (2), the fermentation temperature is 35-37℃.

[0058] As more preferred, in step (2), the fermentation temperature is 37℃, the pH is 7, and the fermentation time is 108 h.

[0059] As more preferred, in step (2), the aeration rate is 2.0vvm, and the stirring speed is 200 rpm.

[0060] As more preferably, in step (2), the final concentration of IPTG is 0.2 mM.

[0061] As a preferred scheme, the method is specifically as follows:

[0062] (1) The E. coli strain QJS-2538 glycerol bacteria are streaked on LB solid medium containing ampicillin and kanamycin, and incubated at 37°C overnight; then a single bacterium is picked and inoculated into a liquid medium, and incubated at 37°C, 220 rpm for 12 hours;

[0063] (2) Seed liquid is obtained by shake flask culture: the bacterial liquid obtained in step (1) is subjected to shake flask culture in a liquid medium, and incubated at 37°C, 220 rpm for 12 hours, to obtain a seed liquid with OD 600 of 3-5;

[0064] (3) Liquid medium is added to the fermentation liquid; after sterilization, the temperature is controlled at 37°C, the pH is controlled at 7.0, and the dissolved oxygen is controlled at 100%; the seed liquid is inoculated into the fermentation tank for culture, the rotation speed is adjusted to 200 rpm, the air input amount is 2vvm, and the dissolved oxygen is controlled at 30%-50% during the fermentation process; in the early stage, the acetic acid salt concentration is controlled at 3-6 g / L by flow addition of acetic acid salt, until the OD 600 reaches 15-20, then IPTG is added to a final concentration of 0.2 mM for induction, and the acetic acid salt concentration is controlled at 5-8 g / L by flow addition of acetic acid salt;

[0065] (4) Samples are taken every 5 hours, and the contents of acetic acid and hydroxysafflor yellow A in the fermentation liquid are detected by HPLC, until the yields of acetic acid and hydroxysafflor yellow A do not increase for 6 consecutive hours, then the fermentation is stopped, and hydroxysafflor yellow A is obtained.

[0066] The present application has the following beneficial effects:

[0067] 1. The present application is to construct a genetically engineered Escherichia coli BL21 by metabolic engineering, knock out phosphoenolpyruvate carboxylase (PPC), L-tyrosine transcriptional regulator (TyrR), anthranilate synthase (TyrE), prephenate dehydratase (PheA) and phenylacetaldehyde dehydrogenase (feaB), block the competition pathway and relieve the metabolic repression; at the same time, overexpress acetyl-CoA synthetase (ACS), isocitrate lyase (aceA), phosphoenolpyruvate carboxykinase (pck), phosphoenolpyruvate synthase (ppsA), fructose-1,6-bisphosphatase (fbp), phosphoglucomutase (pgm), 4-hydroxyphenylacetate hydroxylase (HpaC) and UDP-glucose pyrophosphorylase (galU); and introduce codon-optimized alcohol dehydrogenase (ADH6), phenylpyruvate decarboxylase (ARO10), FAD-dependent 4-hydroxyphenylacetate-3-monooxygenase (HpaB) and glycosyltransferase (RrUGT33), construct acetate metabolism and hydroxyl rhodiolin synthesis pathway; further modify prephenate dehydrogenase (TyrA fbr , M53I / A354V) and 3-deoxy-7-phosphoheptulonate synthase (AroG fbr , D146N) by site-directed mutagenesis technology, strengthen the key node metabolic flux. The finally obtained genetically engineered bacteria QJS-2538 can efficiently synthesize hydroxyl rhodiolin with acetate as the only carbon source under pH 7.0 conditions, the yield in a 5L fermenter reaches 6.3g / L, the conversion rate is 0.45g / g, which significantly reduces the production cost and avoids substrate toxicity. The results show that the genetically engineered bacteria provided by the present application has the industrial application potential for synthesizing hydroxyl rhodiolin by using acetate. The present application provides a new strategy for the industrial production of hydroxyl rhodiolin and the high value utilization of acetate.

[0068] 2. The constructed Escherichia coli strain QJS-2538 can not only efficiently produce hydroxyl rhodiolin by using acetate, but also avoid the toxicity of high-concentration substrates (such as hydroxyl tyrosol) to cells. Compared with traditional chemical synthesis method and biosynthesis method, the present application greatly reduces the production cost, the market price of sodium acetate is about 1.26 yuan / kg, which is much lower than 3500 yuan / kg of hydroxyl tyrosol and 63 yuan / kg of tyrosine, and is more suitable for the large-scale and industrial production of hydroxyl rhodiolin. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 is a metabolic flow chart. DETAILED DESCRIPTION

[0070] The technical solutions of the present application will be further clearly and completely described in combination with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Therefore, all the other embodiments obtained by those skilled in the art based on the embodiments in the present application without creative effort fall within the protection scope of the present application.

[0071] In the embodiments of the present application, the experimental methods used are conventional methods unless otherwise specified; and the materials, reagents and the like used can be purchased through conventional marketing unless otherwise specified.

[0072] In the embodiments of the present application, the gene information of some enzymes is shown in Table 1.

[0073] Table 1

[0074] No. Name NCBI No. No. Name NCBI No. 1 PPC enzyme GeneID:948457 8 pck enzyme GeneID:945667 2 TyrR enzyme GeneID:945879 9 ppsA enzyme GeneID:946209 3 TrpE enzyme GeneID:945846 10 fbp enzyme GeneID:948753 4 PheA enzyme GeneID:947081 11 pgm enzyme GeneID:945271 5 feaB enzyme GeneID:945933 12 HpaC enzyme GenBank:AAR11356.1 6 ACS enzyme GeneID:948572 13 galU enzyme GeneID:945730 7 aceA enzyme GeneID:948517 - - -

[0075] In the embodiments of the present application, the composition of the liquid culture medium is as follows: Na2HPO4·12H2O 15.12 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L, MgSO4·7H2O 0.01 g / L, CaCl2 0.02 g / L, CH3COONa 5 g / L, vitamin B1 0.1% (v / v), trace elements 0.1% (v / v) and antibiotics. The formula of the trace elements is as follows: CuCl2·2H2O 0.01 g / L, H3BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, MnCl2·4H2O 0.03 g / L, ZnSO4·7H2O 0.1 g / L, NiCl2·6H2O 0.02 g / L, Na2MoO4·2H2O 0.03 g / L. The antibiotics are ampicillin 100 μg / mL and kanamycin 50 μg / mL.

[0076] The composition of the solid culture medium is as follows: LB culture medium, agar 20 g / L and antibiotics, wherein the antibiotics are ampicillin 100 μg / mL and kanamycin 50 μg / mL.

[0077] Example 1. Construction of Escherichia coli strain QJS-2538

[0078] (1) Construction of gRNA expression vectors targeting PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme

[0079] The PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme, and feaB enzyme of the E. coli strain BL21 were knocked out by the CRISPR / Cas9 system according to the method of the reference "Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system". First, the pTarget plasmid was used as a template, and pTa-F1-PPC-sgRNA and pTarget-R1, pTa-R1-PPC-sgRNA and pTarget-F1, pTa-F1-TyrR-sgRNA and pTarget-R1, pTa-R1-TyrR-sgRNA and pTarget-F1, pTa-F1-TrpE-sgRNA and pTarget-R1, pTa-R1-TrpE-sgRNA and pTarget-F1, pTa-F1-PheA-sgRNA and pTarget-R1, pTa-R1-PheA-sgRNA and pTarget-F1, pTa-F1-feaB-sgRNA and pTarget-R1, pTa-R1-feaB-sgRNA and pTarget-F1 primers were amplified according to the amplification method described in the PrimeSTAR Max DNA Polymerase (TaKaRa) manual, and then the resulting PPC-P1 and PPC-P2, TyrR-P1 and TyrR-P2, TyrE-P1 and TyrE-P2, PheA-P1 and PheA-P2, feaB-P1 and feaB-P2 fragments were respectively Gibson assembled to obtain a gRNA expression vector with PPC, TyrR, TyrE, PheA, and feaB enzymes.

[0080] The required primers are shown in Table 2.

[0081] Table 2

[0082]

[0083] (2) Construction of TyrA fbr enzyme mutants, AroG fbr enzyme mutants, ACS enzyme, aceA enzyme, pck enzyme, and ppsA enzyme fbr -AroG fbr -acs-aceA-pck-ppsA

[0084] 1) TyrA fbrThe enzyme mutant is based on its TyrA enzyme by mutating the methionine at position 53 to isoleucine and the alanine at position 354 to valine, AroG fbr The enzyme mutant is based on its TyrA enzyme by mutating the aspartic acid at position 146 to asparagine, then by connecting the two genes through an SD sequence (as shown in SEQ ID NO: 38) between the BamHI and EcoRI endonucleases of the vector synthesized directly by GenScript Biotech Corporation into pRSFDuet-1, to obtain pRSFDuet-1-TyrA fbr -AroG fbr Expression vector. Wherein, TyrA fbr Enzyme and AroG fbr The amino acid sequences of the enzymes and AroG are shown in SEQ ID NO: 13 and SEQ ID NO: 14, and the nucleotide sequences are shown in SEQ ID NO: 15 and SEQ ID NO: 16.

[0085] 2) Using the Saccharomyces cerevisiae S288C genome as a template, the ACS fragment, the aceA fragment, the pck fragment and the ppsA fragment were amplified using primers acs-F1 / acs-R1, aceA-F1 / aceA-R1, pck-F1 / pck-R1 and ppsA-F1 / ppsA-R1, respectively, according to the amplification method described in the PrimeSTAR Max DNA Polymerase (TaKaRa) instruction manual. Then, the vector pRSFDuet-1-TyrA fbr -AroG fbr linearized, and the ACS fragment and pRSFDuet-1-TyrA fbr -AroG fbr The linearized fragments were Gibson assembled to obtain pRSFDuet-1-TyrA fbr -AroG fbr -acs vector.

[0086] 3) Using the aceA fragment, the pck fragment and the ppsA fragment as templates, the aceA-pck-ppsA fragment was obtained by overlapping PCR using primers aceA-F1 / ppsA-R1 according to the amplification method described in the PrimeSTAR Max DNA Polymerase (TaKaRa) instruction manual. Then, the vector pRSFDuet-1-TyrA fbr -AroG fbr- linearization of the acs vector, the aceA-pck-ppsA fragment and pRSFDuet-1-TyrA fbr - AroG fbr - linearization of the acs vector, the aceA-pck-ppsA fragment and pRSFDuet-1-TyrA fbr - AroG fbr - acs-aceA-pck-ppsA vector. The sequences of the primers are shown in Table 3.

[0087] Table 3

[0088] SEQ ID NO. Primer Name Primer Sequence (5'-3') SEQ ID NO. Primer Name Primer Sequence (5'-3') 1 acs-F1 SEQ ID NO: 17 7 ppsA-F1 SEQ ID NO: 23 2 acs-R1 SEQ ID NO: 18 8 ppsA-R1 SEQ ID NO: 24 3 aceA-F1 SEQ ID NO: 19 9 aroG-R1 SEQ ID NO: 25 4 aceA-R1 SEQ ID NO: 20 10 pRSFDuet-1-F1 SEQ ID NO: 26 5 pck-F1 SEQ ID NO: 21 11 pRSFDuet-1-F2 SEQ ID NO: 27 6 pck-R1 SEQ ID NO: 22 12 pRSFDuet-1-R2 SEQ ID NO: 28

[0089] (3) Construction of the expression vector pETDuet-1-fbp-pgm-galU-ARO10-ADH6-HpaB-HpaC-RrUGT33 of fbp enzyme, pgm enzyme, galU enzyme, ARO10 enzyme, ADH6 enzyme, HpaB enzyme, HpaC enzyme and RrUGT33 enzyme

[0090] The expression vector pETDuet-1-fbp-pgm-galU-ARO10-ADH6-HpaB-HpaC-RrUGT33 was directly synthesized by GenScript, in which the fbp enzyme, pgm enzyme, galU enzyme, ARO10 enzyme and ADH6 enzyme were sequentially inserted between the BamHI endonuclease and EcoRI endonuclease of the pETDuet-1 vector, the HpaB enzyme, HpaC enzyme and RrUGT33 enzyme were sequentially inserted between the Fsel endonuclease and Xhol endonuclease of the pETDuet-1 vector, and the gene fragments were connected by the SD sequence (SEQ ID NO: 38). The amino acid sequences of the ARO10 enzyme, ADH6 enzyme, HpaB enzyme and RrUGT33 enzyme are shown in SEQ ID NOs: 29-32, and the nucleotide sequences are shown in SEQ ID NOs: 33-36, respectively. The nucleotide sequence of the HpaC enzyme is shown in SEQ ID NO: 37.

[0091] (4) First, the gRNA expression vector targeting the PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme obtained in step (1) was introduced into the competent cells of E. coli BL21 to obtain E. coli BL21 in which the PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme were inactivated; then, the pRSFDuet-1-TyrA fbr - AroG fbr- the acs-aceA-pck-ppsA vector, the pETDuet-1-fbp-pgm-galU-ARO10-ADH6-HpaB-HpaC-RrUGT33 vector in step (3) are transformed into the E. coli BL21 competent cells in which the PPC enzyme, the TyrR enzyme, the TyrE enzyme, the PheA enzyme and the feaB enzyme are inactivated to obtain the E. coli strain QJS-2538.

[0092] Step (4) in the construction of the E. coli BL21 in which the PPC enzyme, the TyrR enzyme, the TyrE enzyme, the PheA enzyme and the feaB enzyme are inactivated, the Cas9 system is activated after the gRNA expression vector is transformed into the S. cerevisiae competent cells in the presence of the gRNA expression vector targeting the PPC enzyme, the TyrR enzyme, the TyrE enzyme, the PheA enzyme and the feaB enzyme to achieve the site-directed editing of the yeast genome and obtain the E. coli BL21 in which the PPC enzyme, the TyrR enzyme, the TyrE enzyme, the PheA enzyme and the feaB enzyme are inactivated. The Cas9 system is the gene editing pCas9 expression plasmid.

[0093] Example 2. Fermenter production of hydroxyl salidroside

[0094] The E. coli strain QJS-2538 is used to produce hydroxyl salidroside by using acetic acid in a 5L fermenter, and the specific steps are as follows:

[0095] (1) Take out one 1.5mL EP tube containing the E. coli strain QJS-2538 glycerol bacteria from the -80℃ refrigerator, streak on the LB solid medium containing ampicillin and kanamycin, and incubate overnight in a 37℃ constant temperature incubator, then pick single bacteria into 6mL liquid medium, and incubate at 37℃ in a shaker at 220rpm for 12h.

[0096] (2) Use one 500mL shake flask to culture the seed liquid, fill 150mL of liquid medium in the shake flask, take 1.5mL of the above cultured 6mL bacterial liquid and add it into the shake flask, then incubate at 37℃ in a shaker at 220rpm for about 12h to obtain the seed liquid OD 600 which is 3-5, and a total of 150mL of seed liquid is obtained.

[0097] (3) Fill 3L of liquid medium in a 5L fermentation liquid, sterilize, control the temperature at 37℃, the pH at 7.0 and the dissolved oxygen at 100%, inoculate 150mL of seed liquid into the 5L fermenter to start the culture, adjust the rotation speed to 200rpm and the gas flow to 2vvm, and control the dissolved oxygen in the fermentation process at 30%-50%. In the early stage, add acetic acid salt to make the acetic acid salt concentration at 3-6g / L until the OD 600When the pH reached 15-20, IPTG was added to a final concentration of 0.2 mM for induction, and the acetic acid concentration was controlled at 5-8 g / L by feeding acetic acid.

[0098] (4) Every 5 hours, the content of acetic acid and hydroxylsalidroside in the fermentation broth was detected by HPLC, and the fermentation was stopped until the yield of acetic acid and hydroxylsalidroside did not increase for 6 consecutive hours.

[0099] After 108 h of fermentation, the yield and conversion rate of hydroxylsalidroside were 6.3 g / L and 0.45 g / g (hydroxylsalidroside / acetic acid), respectively.

Claims

1. A genetically engineered bacterium for synthesizing hydroxysafflor yellow A, characterized in that, The genetically engineered bacteria is obtained by knocking out PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme on the genome of Escherichia coli, overexpressing ACS enzyme, aceA enzyme, pck enzyme, ppsA enzyme, fbp enzyme, pgm enzyme, HpaC enzyme, galU enzyme and mutated TyrA fbr enzyme and AroG fbr enzyme, and introducing ADH6 enzyme, ARO10 enzyme, RrUGT33 enzyme and HpaB enzyme.

2. The genetically engineered bacteria according to claim 1, characterized in that, The TyrA fbr The amino acid sequence of the enzyme is shown as SEQ ID NO: 13, and the nucleotide sequence is shown as SEQ ID NO: 15; the AroG fbr The amino acid sequence of the enzyme is shown as SEQ ID NO: 14, and the nucleotide sequence is shown as SEQ ID NO:

16. 3.The genetically engineered bacteria according to claim 1, characterized in that, The ADH6 enzyme is a codon-optimized ADH6 enzyme from Saccharomyces cerevisiae S288C, the amino acid sequence of which is shown as SEQ ID NO: 30, and the nucleotide sequence of which is shown as SEQ ID NO: 34; The ARO10 enzyme is a codon-optimized ARO10 enzyme from Saccharomyces cerevisiae S288C, the amino acid sequence of which is shown as SEQ ID NO: 29, and the nucleotide sequence of which is shown as SEQ ID NO: 33; The RrUGT33 enzyme is a codon-optimized RrUGT33 enzyme from Rhodiola rosea, the amino acid sequence of which is shown as SEQ ID NO: 32, and the nucleotide sequence of which is shown as SEQ ID NO: 36; The HpaB enzyme is a codon-optimized HpaB enzyme from Pseudomonas aeruginosa, the amino acid sequence of which is shown as SEQ ID NO: 31, and the nucleotide sequence of which is shown as SEQ ID NO:

35. 4.The genetically engineered bacteria according to claim 1, characterized in that, The Escherichia coli is Escherichia coli BL21 strain.

5. The method for preparing the genetically engineered bacterium according to any one of claims 1 to 4, characterized by, The method comprises the following steps: S1: constructing a gRNA expression vector targeting PPC enzyme, TyrR enzyme, TrpE enzyme, PheA enzyme and feaB enzyme; S2: TyrA fbr Enzyme mutant, AroG fbr The enzyme mutant, ACS enzyme, aceA enzyme, pck enzyme and ppsA enzyme are constructed into a vector to obtain a first recombinant expression vector; S3: constructing fbp enzyme, pgm enzyme, galU enzyme, ARO10 enzyme, ADH6 enzyme, HpaB enzyme, HpaC enzyme and RrUGT33 enzyme into a vector to obtain a second recombinant expression vector; S4: introducing the gRNA expression vector obtained in S1 into an Escherichia coli competent cell to obtain an Escherichia coli in which PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme are knocked out; S5: transforming the first recombinant expression vector obtained in S2 and the second recombinant expression vector obtained in S3 into the Escherichia coli in which PPC enzyme, TyrR enzyme, TyrE enzyme, PheA enzyme and feaB enzyme are knocked out to obtain a genetically engineered bacterium.

6. The preparation method according to claim 5, characterized in that In S1, the primers with nucleotide sequences shown as SEQ ID NO: 1-12 are used for amplification; in S2, the primers with nucleotide sequences shown as SEQ ID NO: 17-28 are used for amplification.

7. The preparation method according to claim 5, characterized in that In S2, the vector is pRSFDuet-1 vector; in S3, the vector is pETDuet-1 vector.

8. The genetically engineered bacteria of any one of claims 1 to 4 for use in biosynthesis of hydroxylsalidroside, characterized in that, Acetate is used as the substrate.

9. The method for synthesizing hydroxylsalidrosides by using the genetically engineered bacteria according to claim 1, characterized in that, The genetically engineered bacterium according to any one of claims 1-4 and / or the engineered bacterium constructed by the preparation method according to any one of claims 5-7 is used as a fermentation bacterium, acetate is used as the substrate, and hydroxy rhodioside is produced.

10. The method of claim 9, wherein, The fermentation culture conditions include that the concentration of acetate is 3-8 g / L, the fermentation temperature is 30-37℃, the pH is 5.5-7, the fermentation time is 100-115 h, the aeration amount is 2.0-3.0vvm, the stirring speed is 200-900 rpm, and the dissolved oxygen concentration is 30%-50%.

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