CN107460203B

CN107460203B - Recombinant bacterium for producing salidroside and analogues thereof, construction method and application

Info

Publication number: CN107460203B
Application number: CN201610408741.2A
Authority: CN
Inventors: 赵广荣; 李晓波; 刘雪
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2016-06-03
Filing date: 2016-06-03
Publication date: 2021-06-18
Anticipated expiration: 2036-06-03
Also published as: CN107460203A

Abstract

The invention discloses a recombinant bacterium for producing salidroside and analogues thereof, a construction method and application thereof, wherein the construction method comprises the following steps: synthesizing a keto decarboxylase gene synkdc and a glycosyltransferase gene synyjic, connecting the synthetic ketodecarboxylase gene synkdc and the glycosyltransferase gene synyjic to a vector pRSFDuet-1, and constructing a recombinant vector pSynkdc 1-yjic; dissociating or integrating a T7RNA polymerase gene into a SyBE-002447 chassis strain, and introducing pSynkdc1-yjic to obtain SyBE-218011 and SyBE-218013 strains; directly integrating synkdc and synyjic into SyBE-002447 chromosome, and simultaneously knocking out feaB and ushA to obtain SyBE-218015 strain; the strain of the invention is used for producing salidroside and analogues thereof by fermentation, can solve the problem of sources of salidroside and analogues thereof, and reduces the cost.

Description

Recombinant bacterium for producing salidroside and analogues thereof, construction method and application

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a recombinant bacterium for producing salidroside and analogues thereof and a construction method.

Background

Salidroside is the main effective component of Rhodiola (Rhodiola rosea) of Rhodiola of Crassulaceae, and has good pharmacological activities of resisting oxidation, radiation, fatigue, immunity, altitude hypoxia, etc. The molecular formula is C₁₄H₂₀O₇Molecular weight 300.30, white-like or pale yellow powder. In recent years, research shows that salidroside can be used for various treatments such as cancer, nervous system diseases, cell aging, hypoxia and ischemia of organs such as brain and heart, cognitive impairment, skin pigmentation, metabolism regulation and the like, so that salidroside is widely used in industrial production of medicines, health products, cosmetics and the like.

At present, the main source of salidroside is still wild rhodiola, the amount of salidroside in wild rhodiola is very low, and the extraction of natural salidroside requires a complex process, and the amount of salidroside in the plants of rhodiola sachalinensis and rhodiola crenulata which are most commonly used at present is only 0.5% -0.8%. The cost of the artificially cultivated rhodiola rosea is rather high and the amount of active ingredients is low. In recent years, important improvements are made on relevant theories and practices in aspects of chemical synthesis, biocatalytic (enzymatic) synthesis of salidroside and the like. Although techniques for the chemical synthesis of salidroside and analogues thereof are becoming more mature, most require selective protection, activation or the use of expensive metal catalysts. Therefore, the above methods are not suitable for industrial production.

The biosynthesis of salidroside in plants is divided into 4 stages: the first stage is that the primary metabolite phosphoenolpyruvate and erythrose-4-phosphate form shikimic acid via the shikimic acid pathway; the second stage is to form the aromic acid by shikimic acid through several enzymatic reactions; the third stage is the synthesis of tyrosol from aronic acid; the fourth stage is the formation of salidroside from the combination of glucose and tyrosol. Of these 4 stages, the l-th stage is a metabolic step common to many higher plants and is well-defined, and the reaction mechanisms of the 2 nd and 4 th stages have been explored; with regard to the 3 rd stage biosynthetic pathway from allonic acid to tyrosol, 3 possible pathways have been proposed, namely the phenylpropanoid metabolic pathway, the tyrosine decarboxylation metabolic pathway and the tyrosine transamination metabolic pathway.

In recent years, research has also been carried out to confirm that tyrosine can be directly converted into tyrosol through decarboxylation, reduction and other steps. The last step reaction mechanism of salidroside biosynthesis is also clear, and uridine diphosphate glucosyltransferase (UDP-glucosyltransferase, UDPGT, UGTs) in plants catalyze the synthesis of salidroside by using uridine diphosphate glucose (UDPG) and tyrosol as substrates. Therefore, by using synthetic biology techniques, a completely new biosynthesis pathway is constructed in the engineered microorganisms, and the de novo synthesis of salidroside and analogues thereof is realized.

Disclosure of Invention

The invention aims to provide a recombinant bacterium for producing salidroside and analogues thereof, which can utilize a keto decarboxylase gene kdc in Pichia pastoris GS115 and a glycosyl transferase gene yjic in Bacillus licheniformis to synthesize the salidroside and analogues thereof by using biomass carbon sources such as glucose.

The second purpose of the invention is to provide a construction method of a recombinant bacterium for producing salidroside and analogues thereof.

The third purpose of the invention is to provide a method for preparing salidroside and analogues thereof by fermenting recombinant bacteria for producing salidroside and analogues thereof.

The technical scheme of the invention is summarized as follows:

(1) artificially and totally synthesizing a keto decarboxylase gene synkdc, wherein the keto decarboxylase gene synkdc is shown as SEQ ID No. 03; artificially and totally synthesizing a glycosyltransferase gene synyjic, wherein the glycosyltransferase gene synyjic is shown as SEQ ID No. 04;

(2) connecting the synkdc gene to the downstream of a first promoter of an expression vector pRSFDuet-1 through enzyme digestion, and screening positive clones to obtain a recombinant vector pSynkdc 1;

(3) connecting the synyjic gene to the downstream of a second promoter of pSynkdc1, and screening positive clones to obtain a recombinant vector pSynkdc 1-yjic;

(4) cloning from Escherichia coli BL21(DE3) to obtain T7RNA polymerase gene, connecting to vector pYSC1, screening positive clone to obtain recombinant vector pYBH 1;

(5) the method is carried out in one of the following three ways:

the first method is as follows:

cloning T7RNA polymerase gene from Escherichia coli BL21(DE3) and integrating the T7RNA polymerase gene on chromosome of SyBE-002447 chassis strain by lambda Red homologous recombination technology to obtain SyBE-002447(DE3) strain;

secondly, the recombinant vector pSynkdc1-yjic is transformed into escherichia coli SyBE-002447(DE3) to obtain a SyBE-218011 strain;

the second method comprises the following steps:

firstly, transforming a recombinant vector pYBH1 into escherichia coli SyBE-002447 to obtain a SyBE-218012 strain;

secondly, the recombinant vector pSynkdc1-yjic is transformed into escherichia coli SyBE-218012 to obtain a SyBE-218013 strain;

the third method comprises the following steps:

firstly, integrating a synkdc gene into escherichia coli SyBE-002447(DE3) through a lambda Red homologous recombination technology, and knocking out a gene feaB to obtain a SyBE-218014 strain;

firstly, the synyjic gene is integrated into the Escherichia coli SyBE-218014 strain through a lambda Red homologous recombination technology, and the ushA gene is knocked out to obtain the SyBE-218015 strain.

The recombinant bacterium SyBE-218011 strain, SyBE-218013 strain or SyBE-218015 strain for producing salidroside and analogues thereof constructed by the method.

The method for preparing salidroside and analogues thereof by recombinant bacteria producing salidroside and analogues thereof comprises the following steps:

inoculating recombinant bacteria SyBE-218011, SyBE-218013 or SyBE-218015 in LB culture medium, culturing at 37 deg.C and 220rpm with shaking for 2-4h at OD₆₀₀When the concentration of glucose reaches 0.8-1.0, transferring to M9 culture medium containing glucose or culture medium for enzymatic hydrolysis of salidroside plant raw material, fermenting, and supplementing 5g/L glucose when the concentration of glucose is lower than 0.5g/L, and continuously synthesizing salidroside and its analogues.

The invention has the advantages that:

salidroside can be used for treating cancer, nervous system diseases, cell aging, etc., so that Salidroside can be widely used in industrial production of cosmetics, food, health products, medicine, etc. The invention uses engineering colibacillus and glucose as carbon source, and utilizes different strategies of free induction expression and integrated expression to ferment and produce salidroside and analogues thereof. The invention can solve the problem of sources of salidroside and analogues thereof, simultaneously reduces the production cost to the maximum extent and is beneficial to industrial production.

Drawings

FIG. 1 shows the synthesis route of salidroside and its analogues (phenethyl alcohol-beta-D-glucopyranoside).

FIG. 2 is an HPLC chromatogram verified for salidroside and analogues thereof.

(a) The detection spectrum of the fermentation broth of the recombinant bacterium SyBE-218011, (b) the detection spectrum of the fermentation broth of the recombinant bacterium SyBE-218013, and (c) the detection spectrum of the fermentation broth of the recombinant bacterium SyBE-218015.

Detailed Description

The ketodecarboxylase gene kdc in Pichia pastoris GS115 and the glycosyltransferase gene yjic in Bacillus licheniformis are utilized to realize the synthesis of salidroside and analogues thereof in engineering Escherichia coli by taking glucose as a biomass carbon source, so that the production cost is minimized.

The original Chassis E.coli is a high tyrosine-producing strain, named SyBE-002447, under the taxonomic name: escherichia coli (Escherichia coli) is currently preserved in the China general microbiological culture Collection center (CGMCC), and the registration number of the preservation center is CGMCC No. 7962. The preservation time is 7 months and 22 days in 2013, and the address is microbial research institute of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, North Cheng.

Coli strains e.coli BL21(DE3) and e.coli DH5 α used in the present invention were purchased from beijing hologold biotechnology limited.

The expression vector pRSFDuet-1 used in the present invention was purchased from Novagen.

The pKD46 plasmid and the pCP20 plasmid used in the present invention were purchased from proetin Biotechnology (Beijing) Co., Ltd.

The LB medium consisted of: 10g/L NaCl, 10g/L peptone and 5g/L yeast powder, the balance being water, sterilizing at 121 ℃ under 0.1MPa for 20 min.

The composition of the glucose-containing M9 medium was: 0.5g/L NaCl, 1.0g/L NH₄Cl、3.0g/L KH₂PO4、17.1g/L Na₂HPO₄·12H₂O, 0.025% yeast powder, and the balance water, and sterilizing the culture medium at 121 deg.C under 0.1Mpa for 20 min. After sterilization, the final concentration of 5mM MgSO 5 was added to the membrane₄、0.1mM CaCl₂And sterilized 5g/L glucose.

The culture medium containing salidroside plant raw material enzymatic hydrolysate: pulverizing rhodiola into powder, wherein the powder comprises the following components: water 1: 15, adding cellulase, pectinase and hemicellulase for hydrolysis, and 0.1% KH₂PO4、0.1％Na₂HPO₄·12H₂O and 0.1% NH₄Cl, medium was sterilized at 121 ℃ under 0.1MPa for 20 min.

The present invention will be further described with reference to the following examples.

The biosynthesis pathway of salidroside designed by the invention belongs to the shikimic acid pathway, and the detailed synthesis pathway is as follows: glucose or other biomass is used as a carbon source, and p-hydroxyphenylpyruvic acid is synthesized through a shikimic acid pathway. The synthesis of 4-hydroxyphenylacetaldehyde from p-hydroxyphenylpyruvate under the catalysis of keto decarboxylase (synkdc), the production of 4-hydroxyphenylethanol (tyrosol) from 4-hydroxyphenylacetaldehyde under the catalysis of escherichia coli endogenous ethanol dehydrogenase (adh), and the synthesis of salidroside and its analogues (phenethyl alcohol-beta-D-glucopyranoside) from tyrosol and UDP-glucose donor under the catalysis of glycosyl transferase (synyjic) are shown in the schematic diagram of the biosynthesis pathway in FIG. 1.

Example 1 design of the ketodecarboxylase Gene synkdc and the glycosyltransferase Gene synyjic

Preferably, the amino acid sequences of the Pichia pastoris GS115 ketodecarboxylase gene and the Bacillus licheniformis glycosyltransferase gene are respectively shown as SEQ ID No.01 and SEQ ID No.02 in the sequence table. The codon preference was optimized in E.coli using JCAT online codon optimization software (http:// www.jcat.de) in combination with the OPTIMIZER online codon optimization tool (http:// genes. urv. es/OPTIMIZER /), to design the full-length synkdc gene and synyjic gene as shown in sequence listings SEQ ID No.03 and SEQ ID No.04, respectively.

Example 2 lambda-red homologous recombination method integrates T7RNA polymerase into the chromosome of the Chassis strain SyBE-002447.

The detailed steps of the construction process of the chassis strain integrating T7RNA polymerase on the chromosome of the chassis strain SyBE-002447 are as follows:

1. design primers T7-RNA F and T7-RNA R with sequences as SEQ ID NO.07 and SEQ ID NO.08 respectively clone T7RNA polymerase gene from Escherichia coli BL21(DE3), wherein the gene sequence of the T7RNA polymerase is as SEQ ID NO. 05.

2. The primers T7F and T7R, T7-Chl F and T7-Chl R are respectively shown in sequence tables SEQ ID NO.09, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, and the overlap extension PCR is used for preparing the T7RNA polymerase fragment with chloramphenicol resistance.

3. Introducing pKD46 plasmid into strain SyBE-002447 to obtain SyBE-002447/pKD46, inoculating activated strain SyBE-002447/pKD46 into 10ml LB liquid culture medium, culturing at 30 deg.C and 200rpm to OD₆₀₀Is 0.4-0.6. L-arabinose was added to the mixture at a final concentration of 10mM, and the culture was continued for 3 hours. The cells were collected by centrifugation at 4000rpm for 8min at 4 ℃. The supernatant was discarded, and cells were washed 2 times with ice-chilled 10% glycerol to prepare electroporation competent cells. mu.L of the resistant fragment was added to 100. mu.L of the electroporation competent cells, and gently swirled to mix well. The mixture was added to a 2mm ice-chilled cuvette and shocked at 2.5KV for 4-6 ms. Then, 1mL of LB medium was added, and after 3 hours of recovery at 37 ℃ the corresponding resistant plates were spread and cultured overnight at 42 ℃.

4. And selecting a single colony which is verified to be correct by PCR, carrying out passage for 3 times on a chloramphenicol resistant plate at 42 ℃, verifying that pKD46 is deleted on an ampicillin resistant plate, and carrying out culture and preservation at 37 ℃ to obtain a strain integrating a T7RNA polymerase fragment into the chromosome of the chassis strain SyBE-002447.

5. Competent cells were prepared from the recombinant bacteria obtained in step 2, and the plasmid pCP20 was electrically transformed in the same manner and cultured overnight at 30 ℃. Selecting single colony, streaking by using a non-resistant plate, carrying out passage for 3 times at 42 ℃, screening strains with chloramphenicol and ampicillin resistance deletion, and culturing and preserving at 37 ℃ to obtain an optimized chassis strain SyBE-002447(DE3) successfully integrated without trace.

EXAMPLE 3 construction of recombinant expression vector pSynkdc1-yjic

After EcoRI and PstI enzyme cutting sites positioned at the downstream of a first promoter of an expression vector pRSFDuet-1 are respectively added to two ends of a synkdc gene fragment obtained by chemical total synthesis through a PCR method, FastDiget endonuclease EcoRI and PstI are used for enzyme cutting, and the reaction system is as follows: 5 μ L of 10 × FD buffer, 2.5 μ L of EcoRI, 2.5 μ L of PstI, 30 μ L of synkdc gene fragment and 10 μ L of ultrapure water. The reaction conditions are as follows: 37 ℃ for 2 h. Adding 250 mu L of binging Buffer solution into 50 mu L of enzyme digestion product by using a PCR purification kit, uniformly mixing, adding into an adsorption column, standing for one minute, centrifuging for 1 minute at 10,000 g, and removing effluent. 650. mu.L of Wash Buffer,10,000 g, was added and centrifuged for 1 min, and the effluent was discarded. Centrifuge at 10,000 g for 2 minutes to remove the residual Wash Buffer. The adsorption column was placed in a clean centrifuge tube, 30. mu.L of Elution Buffer (which was preheated in advance in a 65 ℃ water bath) was added to the center of the column, and the column was allowed to stand at room temperature for 1 minute, centrifuged at 10,000 g for 1 minute, and the cleaved synkdc gene fragment was eluted. The pRSFDuet-1 plasmid was similarly digested with Fastdigest endonucleases EcoRI and PstI, and purified and recovered with a PCR purification kit. The digested synkdc gene fragment and pRSFDuet-1 plasmid were ligated. The reaction system is as follows: mu.L of 10 × T4DNA Ligase Buffer, 1. mu. L T4DNA Ligase, 6. mu.L of the digested synkdc gene fragment and 2. mu.L of the digested pRSF-Duet1 plasmid. The reaction conditions are as follows: 22 ℃ for 0.5 h. And transforming competent cells E.coli DH5 alpha after enzyme digestion and connection, screening positive clone plasmids by colony PCR, and verifying sequencing to obtain a plasmid pSynkdc1 in which the synkdc gene is connected to the downstream of the first promoter of the expression vector pRSFDuet-1.

After XhoI and BglII enzyme cutting sites positioned at the downstream of a second promoter of an expression vector pRSFDuet-1 are respectively added at two ends of a synyjic gene fragment obtained by chemical total synthesis through a PCR method, the synyjic gene fragment and pSynkdc1 plasmid are subjected to double enzyme cutting by Fastdigest endonuclease XhoI and BglII respectively, then the synyjic gene is connected to the downstream of the second promoter of a pSynkdc1 vector through DNA ligase, and an enzyme cutting connection reaction system and reaction conditions are constructed according to the pSynkdc1 vector. And (3) transforming competent cells E.coli DH5 alpha after enzyme digestion and connection, screening positive clones by colony PCR to obtain a recombinant vector pSynkdc1-yjic, and sequencing and verifying.

Example 4 transformation of recombinant expression vector pSynkdc1-yjic into E.coli Chassis Strain SyBE-002447(DE3)

The detailed construction steps of the recombinant vector pSynkdc1-yjic transformed into the Escherichia coli SyBE-002447(DE3) are as follows:

1. the activated Escherichia coli strain SyBE-002447(DE3) was inoculated in 100. mu.L of 10ml LB medium at 37 ℃ and 220rpm and cultured to OD₆₀₀Transferring into 10ml centrifuge tube when the concentration is 0.8-1.0, and precoolingCentrifuging at 4500rpm/min for 5min in a 4 deg.C centrifuge, removing supernatant, and collecting thallus;

2. washing the thallus with 5ml of precooled sterilized 10% glycerol, centrifuging at 4500rpm/min in a precooled 4 ℃ centrifuge for 5min, removing supernatant, collecting thallus, and repeatedly washing for three times;

3. pouring the supernatant as much as possible, adding 200 μ L of 10% glycerol to resuspend the thallus, making into SyBE-002447(DE3) competent cells, and subpackaging into two tubes;

4. mu.L of the pSynkdc1-yjic plasmid 2.5 constructed in example 3 was added to 100. mu.L of the electroporation competent cells, and the mixture was gently swirled and mixed. The mixture was added to ice pre-cooled 2mm electroporation cuvettes separately and shocked at 2.5KV for 4-6 ms. Rapidly and respectively adding 1mL of LB culture medium, recovering for 2h at 37 ℃, coating a plate with corresponding resistance, and culturing overnight;

5. selecting a positive transformant verified by colony PCR to be cultured in 5ml LB culture medium overnight to obtain a recombinant strain SyBE-218011, and storing the strain;

EXAMPLE 5 construction of recombinant expression vector pYBH1

1. Primers T7Eco F and T7Pst R are designed, and T7RNA polymerase genes are cloned from Escherichia coli BL21(DE3) as shown in sequence tables SEQ ID NO.13 and SEQ ID NO.14 respectively.

2. The PCR product and the pYSC1 vector are respectively subjected to enzyme digestion connection by using Fastdigest endonucleases EcoR1 and Pst1 (the enzyme digestion connection reaction system and the reaction conditions refer to example 3), the enzyme digestion connection product is transformed into a competent cell E.coli DH5 alpha, a colony PCR is used for screening a positive clone extraction plasmid, and sequencing verification is carried out to obtain an expression auxiliary plasmid pYBH1 connected to the pYSC1 vector.

EXAMPLE 6 transformation of expression helper plasmid pYBH1 into E.coli Chassis Strain SyBE-002447

Detailed construction procedure for transforming expression helper plasmid pYBH1 into Escherichia coli SyBE-002447 referring to example 4, recombinant strain SyBE-218012 is obtained, and the strain is preserved.

Example 7 transformation of recombinant expression vector pSynkdc1-yjic into E.coli recombinant Strain SyBE-218012

Detailed construction procedure for transformation of recombinant expression vector pSynkdc1-yjic into E.coli recombinant strain SyBE-218012 referring to example 4, recombinant strain SyBE-218013 was obtained and the strain was preserved.

Example 8 λ -red homologous recombination method knockout of the feaB gene and simultaneous integration of the synkdc gene into the chromosome of the Chassis strain SyBE-002447(DE 3).

The detailed steps of the chassis strain construction process for knocking out the feaB gene and simultaneously integrating the synkdc gene on the chromosome of the chassis strain SyBE-002447(DE3) are as follows:

1. primers KdcF and Kdc R, and Kan F and Kan R have the sequences shown in sequence tables SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively, and overlap extension PCR is performed to prepare Kdc-Kan fragments with Kan resistance.

2. Introducing pKD46 plasmid into strain SyBE-002447(DE3) to obtain SyBE-002447(DE3)/pKD46, inoculating activated strain SyBE-002447(DE3)/pKD46 into 10ml LB liquid culture medium, culturing at 30 deg.C and 200rpm to OD₆₀₀Is 0.4-0.6. Adding L-arabinose with final concentration of 10mM, continuing to culture for 3h, centrifuging at 4000rpm at 4 ℃ for 8min, and collecting cells. The supernatant was discarded, and cells were washed 2 times with ice-chilled 10% glycerol to prepare electroporation competent cells. Add 7.5. mu.L of the resistant fragment to 100. mu.L of the electroporation competent cells, mix by gentle rotation. The mixture was added to a 2mm ice-chilled cuvette and shocked at 2.5KV for 4-6 ms. Then, 1mL of LB medium was added, and after recovery at 37 ℃ for 2 hours, the corresponding resistant plates were spread and cultured overnight at 42 ℃.

3. And selecting a single colony which is verified to be correct by PCR, carrying out passage 3 times on a kanamycin-resistant plate at 42 ℃, verifying that pKD46 is deleted by the ampicillin-resistant plate, and carrying out culture and preservation at 37 ℃ to obtain a strain which is knocked out of the feaB gene and integrates the synkdc gene into the chromosome of a chassis strain SyBE-002447(DE 3).

4. Competent cells were prepared from the recombinant bacteria obtained in step 2, and the plasmid pCP20 was electrically transformed in the same manner and cultured overnight at 30 ℃. Selecting a single colony, streaking a non-resistant plate, carrying out passage for 3 times at 42 ℃, screening kanamycin and ampicillin resistant deletion strains, and culturing and preserving at 37 ℃ to obtain the successfully traceless integrated optimized chassis strain SyBE-218014.

Example 9 lambda-red homologous recombination method to knock out ushA gene and simultaneously integrate synyjic gene into chromosome of E.coli Chassis strain SyBE-218014.

The detailed steps of the construction process of the strain for knocking out the ushA gene and simultaneously integrating the synyjic gene on the chromosome of the escherichia coli chassis strain SyBE-218014 are as follows:

1. the primer YjicF and YjicR, the Yjic-Kan F and the Yjic-Kan R are respectively shown in the sequence tables SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, and the Yjic-Kan fragment with Kan resistance is prepared by overlap extension PCR.

2. The plasmid pKD46 was introduced into E.coli SyBE-218014 to obtain SyBE-218014/pKD 46. Inoculating activated strain SyBE-218014/pKD46 into 10ml LB liquid medium, culturing at 30 deg.C and 200rpm to OD₆₀₀Is 0.4-0.6. Adding L-arabinose with final concentration of 10mM, continuing to culture for 3h, centrifuging at 4000rpm at 4 ℃ for 8min, and collecting cells. The supernatant was discarded, and cells were washed 2 times with ice-chilled 10% glycerol to prepare electroporation competent cells. 7.5. mu.L of the integration resistant fragment Yjic-Kan fragment is taken and added into 100. mu.L of the electroporation competent cells, and the mixture is gently rotated and mixed evenly. The mixture was added to a 2mm ice-chilled cuvette and shocked at 2.5KV for 4-6 ms. Then, 1mL of LB medium was added, and after recovery at 37 ℃ for 2 hours, the corresponding resistant plates were spread and cultured overnight at 42 ℃.

3. A single colony which is verified to be correct by PCR is picked, and is passaged for 3 times on a kanamycin-resistant plate at 42 ℃, and the kanamycin-resistant plate verifies that pKD46 is deleted, and is cultured and preserved at 37 ℃ to obtain a strain integrating yjic genes into the chromosome of the chassis strain SyBE-002447(DE 3).

4. Competent cells were prepared from the recombinant bacteria obtained in step 5, and the plasmid pCP20 was electrically transformed in the same manner and cultured overnight at 30 ℃. Selecting single colony, streaking by using a non-resistant plate, carrying out passage for 3 times at 42 ℃, screening kanamycin and ampicillin resistant deletion strains, and culturing and preserving at 37 ℃ to obtain a chassis strain SyBE-218015 successfully integrated without trace.

Example 10 fermentation and detection of recombinant bacteria SyBE-218011, SyBE-218013 and SyBE-218015

Recombinant strains SyBE-218011, SyBE-218013 and SyBE-218015 strainsThe cells were inoculated into 5ml of LB medium and cultured overnight. The overnight-cultured broth was then transferred into a 250ml shake flask containing 50ml LB medium, starting OD₆₀₀Incubated at about 0.1, 37 ℃ for 3h at 220rpm, OD₆₀₀When the concentration of glucose reaches 0.8, transferring the strain to M9 culture medium containing glucose, fermenting at 30 ℃ and 200rpm, supplementing 5g/L glucose when the concentration of glucose is lower than 0.5g/L, and continuously fermenting and culturing for 72 hours.

Taking 1ml of fermentation liquor at different time points in the fermentation process, then centrifuging at 12000r/min for 10 minutes, taking supernatant, filtering with a 0.22 mu m microporous filter membrane, and then carrying out High Performance Liquid Chromatography (HPLC) system detection. The chromatographic conditions were as follows: a C18 (4.6X 250mm) chromatography column; the mobile phase is 20% methanol, 80% ultrapure water solution and 0.1% formic acid; the flow rate is 1 mL/min; the sample volume is 20 mu L; column temperature room temperature; and an ultraviolet detector with the detection wavelength of 280 nm.

Shown in FIG. 2 are HPLC profiles of product verification in fermentation liquid samples of SyBE-218011, SyBE-218013 and SyBE-218015, respectively.

Continuously fermenting for 72h, wherein the SyBE-218011 strain generates salidroside and analogues thereof phenethyl alcohol-beta-D-glucopyranoside, the SyBE-218012 strain generates salidroside and analogues thereof phenethyl alcohol-beta-D-glucopyranoside, and the SyBE-218014 strain generates salidroside and analogues thereof phenethyl alcohol-beta-D-glucopyranoside.

Example 11 fermentation and detection of recombinant bacteria SyBE-218011, SyBE-218013 and SyBE-218015

Respectively inoculating recombinant strains SyBE-218011, SyBE-218013 and SyBE-218015 into 5ml of LB culture medium for overnight culture;

the overnight-cultured broth was then transferred into a 250ml shake flask containing 50ml LB medium, starting OD₆₀₀Incubated at about 0.1, 37 ℃ for 2h at 220rpm, OD₆₀₀When the concentration of glucose reaches 0.9, transferring the strain to M9 culture medium containing glucose, fermenting at 30 ℃ and 200rpm, supplementing 5g/L glucose when the concentration of glucose is lower than 0.5g/L, and continuously fermenting and culturing for 72 hours.

The results of the test were similar to those of example 8.

Example 12 fermentation and detection of recombinant bacteria SyBE-218011, SyBE-218013 and SyBE-218015

the overnight-cultured broth was then transferred into a 250ml shake flask containing 50ml LB medium, starting OD₆₀₀Cultured at 220rpm for 4h at about 0.1, 37 ℃ and OD₆₀₀When the concentration of glucose reaches 1.0, transferring the strain to M9 culture medium containing glucose, fermenting at 30 ℃ and 200rpm, supplementing 5g/L glucose when the concentration of glucose is lower than 0.5g/L, and continuously fermenting and culturing for 72 hours.

The results of the test were similar to those of example 8.

Example 13 fermentation assays of recombinant bacteria SyBE-218011, SyBE-218012, and SyBE-218015

Recombinant strains SyBE-218011, SyBE-218012 and SyBE-218015 were inoculated into 5ml of LB medium, respectively, and cultured overnight.

Transfer the overnight cultured broth into a 250ml shake flask containing 50ml LB medium, initial OD₆₀₀Cultured at 220rpm for 4h at about 0.1, 37 ℃ and OD₆₀₀When reaching 1.0, adding inducer isopropyl thiogalactoside to make final concentration 0.5mM, inducing at 30 deg.C and 220rpm for 3 hr, transferring into culture medium containing salidroside plant material enzyme hydrolysis, and fermenting.

The results of the test were similar to those of example 8.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

Claims

1. A construction method of a recombinant bacterium for producing salidroside and analogues thereof is characterized by comprising the following steps:

(4) cloning a T7RNA polymerase gene from escherichia coli BL21(DE3) bacteria, connecting the gene to a vector pYSC1, screening positive clones to obtain a recombinant vector pYBH1, wherein the sequence of the vector pYSC1 is shown as SEQ ID No. 05;

(5) the method is carried out in one of the following three ways:

the first method is as follows:

cloning a T7RNA polymerase gene from escherichia coli BL21(DE3) bacteria, and integrating the gene on a chromosome of a SyBE-002447 chassis strain through a lambda Red homologous recombination technology to obtain a SyBE-002447(DE3) strain;

the second method comprises the following steps:

the third method comprises the following steps:

integrating synyjic genes into the Escherichia coli SyBE-218014 strain through a lambda Red homologous recombination technology, and knocking out the ushA gene to obtain a SyBE-218015 strain;

the salidroside analogue is phenethyl alcohol-beta-D-glucopyranoside.

2. Recombinant bacteria SyBE-218011, SyBE-218013, or SyBE-218015 for producing salidroside and analogs thereof constructed by the method of claim 1.

3. The method for preparing salidroside and analogues thereof by recombinant bacteria producing salidroside and analogues thereof is characterized by comprising the following steps:

the recombinant bacterium SyBE-218011, SyBE-218013 or SyBE-218015 of claim 2 is inoculated in LB culture medium, cultured at 37 ℃ and 220rpm with shaking for 2-4h, and cultured at OD₆₀₀When the concentration of glucose reaches 0.8-1.0, transferring to M9 culture medium containing glucose or culture medium for enzymatic hydrolysis of salidroside plant raw material, fermenting, and supplementing 5g/L glucose when the concentration of glucose is lower than 0.5g/L, and continuously synthesizing salidroside and its analogues.