CN108998460B - Biological production method of salidroside - Google Patents

Abstract

The invention discloses a biological production method of salidroside, which comprises a construction method and a fermentation method of recombinant bacteria for producing salidroside. The construction method of the salidroside recombinant strain comprises the following steps: and (3) synthesizing a keto decarboxylase gene skdc with a trc promoter and a glycosyltransferase gene sugt2 with a tac promoter in a total chemical way, and integrating the skdc gene and the sugt2 gene into a SyBE-002447 chromosome to obtain the SDR1 strain. Taking glucose as a precursor, fermenting the SDR1 strain to produce salidroside. The invention uses the engineering escherichia coli to produce the salidroside, can solve the problem of the source of the salidroside and reduce the cost.

Description

Biological production method of salidroside

Technical Field

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

Background

Salidroside (salidroside) with molecular formula of C₁₄H₂₀O₇And the molecular weight is 300.30. Salidroside has good pharmacological activities of resisting altitude stress, oxidation, radiation, fatigue and the like, and is widely applied to cerebral ischemia, fatigue, anoxia and neurodegenerative diseases by Asian Europe countries.

Salidroside is usually extracted from the roots of 3-5 years old wild Rhodiola rosea (Rhodiola), which is costly. Because the rhodiola rosea is distributed in the area with the altitude of more than 2500-5500 meters in the northern hemisphere, such as the Altai mountain and the Himalayan area, and grows and slows down. The synthesis of salidroside by chemical methods requires selective protection, activation or the use of expensive metal catalysts, which is not conducive to industrial production.

Disclosure of Invention

The invention utilizes a ketodecarboxylase gene and a glycosyl transferase gene to realize the synthesis of salidroside by biomass carbon sources such as glucose and the like in engineering escherichia coli, thereby minimizing the production cost.

The second purpose of the invention is to provide a construction method of recombinant bacteria for producing salidroside.

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

The technical scheme of the invention is summarized as follows:

the construction method of the recombinant bacterium for producing salidroside is characterized by comprising the following steps:

(1) artificially and totally synthesizing a keto decarboxylase gene skdc with a trc promoter, wherein the sequence is shown as SEQ ID No. 1; artificially and totally synthesizing a glycosyltransferase gene sugt2 with a tac promoter, wherein the sequence is shown as SEQ ID No. 2;

(2) integrating the skdc gene with the trc promoter into escherichia coli SyBE-002447 through a lambda-red homologous recombination technology, and knocking out a feaB gene to obtain a TRS1 strain;

(3) integrating the sugt2 gene with trc promoter into the escherichia coli TRS1 strain through lambda-red homologous recombination technology, and knocking out lacI gene to obtain SDR1 strain.

The recombinant strain SDR1 for producing salidroside is constructed according to the method.

The method for preparing salidroside by using recombinant bacteria SDR1 for producing salidroside is characterized by comprising the following steps:

inoculating recombinant strain SDR1 into LB culture medium, culturing at 37 deg.C and 220rpm under shaking for 2-4 hr, and culturing at OD₆₀₀When the concentration reaches 0.8-1.0, transferring to M9 culture medium containing glucose, and fermenting for 36h to produce salidroside.

The invention has the advantages that:

salidroside has various pharmacological activities of resisting altitude stress, resisting oxidation, protecting heart, etc., and can be used in industrial production of medicine, food, cosmetic, etc. The invention uses engineering colibacillus and glucose as carbon source, and utilizes constitutive expression fermentation to produce salidroside, and the microbial synthesis cost is low and the yield is high. The invention can solve the problem of the source of salidroside, simultaneously reduces the production cost to the maximum extent and is beneficial to industrial production.

Detailed Description

The production cost minimization of the salidroside synthesis by taking glucose as a biomass carbon source is realized in engineering escherichia coli by utilizing a keto decarboxylase gene skdc and a glycosyl transferase gene sugt 2.

The original Chassis E.coli is a high tyrosine-producing strain, named SyBE-002447, under the taxonomic name: escherichia coli (Escherichia coli). Now, the culture is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, 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.

The pKD46 plasmid used in the present invention was purchased from proetin Biotechnology (Beijing) Inc.

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 10g/L glucose.

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

The invention designs an artificial biosynthesis way of salidroside, and the detailed synthesis process is as follows: glucose or other biomass is used as a carbon source, and the p-hydroxyphenylpyruvic acid is synthesized through a tyrosine pathway. The method is characterized in that p-hydroxyphenylpyruvate is catalyzed by keto decarboxylase (Skdc) to synthesize 4-hydroxyphenylacetaldehyde, the 4-hydroxyphenylacetaldehyde is catalyzed by escherichia coli endogenous alcohol dehydrogenase (Adh) to generate 4-hydroxyphenylethanol (tyrosol), and the tyrosol is combined with UDP-glucose by glycosyltransferase (Sugt2) to synthesize salidroside.

Example 1 design of expression cassettes for the ketodecarboxylase gene skdc and the glycosyltransferase gene sugt2

The preferred ketodecarboxylase gene is optimized for codon preference 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 a full-length skdc gene, and a trc promoter is added in front of the gene to construct a skdc expression cassette, the synthetic sequence is shown in SEQ ID No. 1.

The glycosyltransferase gene is optimized by using JCAT online codon optimization software (http:// www.jcat.de) and an OPTIMIZER online codon optimization tool (http:// genes. urv. es/OPTIMIZER /), codon preference of Escherichia coli is optimized, a full-length sugt2 gene is designed, a trc promoter is added in front of the gene to form a sugt2 expression cassette, and the synthetic sequence is shown as SEQ ID No. 2.

Example 2 λ -red homologous recombination method knockdown of the feaB gene while integrating the skdc gene on chromosome of Chassis strain SyBE-002447.

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

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

1. preparation of integration fragment: KC fragment was prepared by PCR using KF (SEQ ID NO.3) as the upstream primer, KR (SEQ ID NO.4) as the downstream primer, SEQ ID NO.1 as the template, and Phanta Max Super-Fidelity DNA polymerase. The PCR conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃. And purifying and recovering the obtained KC fragment by using a PCR product recovery kit.

2. Homologous recombination: 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. 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 KC fragment 7.5. mu.L to 100. mu.L of 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 thawing at 37 ℃ for 2 hours, the plate was spread and cultured overnight at 37 ℃.

3. Screening of integrated strains: a single colony is picked, TF (SEQ ID NO.5) is used as an upper guide, TR (SEQ ID NO.6) is used as a lower guide, and colony PCR verification is carried out by adopting Taq DNA polymerase. When the size of the PCR band is about 1900bp, the strain TRS1/pKD46 is obtained by knocking out the feaB gene and integrating the skdc gene on the chromosome of the chassis strain SyBE-002447. Colony PCR conditions were pre-denaturation: 97 deg.C, 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1.5min, and 25 cycles; extending for 5min at 72 ℃; storing at 4 ℃.

4. Deletion pKD 46: single colonies that were PCR verified to be correct were picked and serially passaged 3 times on antibiotic-free plates at 42 ℃. Inoculating on an ampicillin resistant plate and a non-resistant plate simultaneously, wherein the ampicillin resistant plate does not grow, and a colony growing on the non-resistant plate is a pKD46 deletion strain, namely obtaining a strain TRS1 which knocks out the feaB gene and integrates the skdc gene on the chromosome of a chassis strain SyBE-002447. After liquid LB culture at 37 ℃ glycerol was stored.

Example 3 lambda-red homologous recombination method knockdown lacI gene and integration of sugt2 gene into chromosome TRS1 of E.coli chassis strain.

The detailed steps of the strain construction process of knocking out lacI gene and simultaneously integrating sugt2 gene on chromosome of escherichia coli chassis strain TRS1 are as follows:

1. preparation of integration fragment: UF is used as an upstream primer (SEQ ID NO.7), UR is used as a downstream primer (sequence SEQ ID NO.8), and SEQ ID NO.2 is used as a template to prepare a UC fragment by PCR. The PCR reaction condition is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃. And purifying and recovering the UC fragment by using a PCR product recovery kit.

2. Homologous recombination: inoculating activated strain TRS1/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. Taking 7.5 mu L of UC fragments, adding the UC fragments into 100 mu L of electroporation competent cells, and gently rotating and mixing the UC fragments evenly. Adding the mixture into a 2mm ice pre-cooling electric shock cupAnd middle 2.5KV electric shock for 4-6 ms. Then, 1mL of LB medium was added, and after thawing at 37 ℃ for 2 hours, the plate was spread and cultured overnight.

3. Screening of the integration strains: a single colony is picked, IF (20bp lacI upstream homology arm, SEQ ID NO.9) is used as an upper guide, IR (20bp sugt2 downstream primer, SEQ ID NO.10) is used as a lower guide, and Taq DNA polymerase is adopted for colony PCR verification. When the size of the PCR band is about 1500bp, the strain SDR1/pKD46 integrating the sugt2 gene on the chromosome of the chassis strain TRS1 is obtained.

4. Deletion pKD 46: the correct single colony SDR1/pKD46 was verified by PCR and passaged 3 times at 42 ℃ without anti-plates. Inoculating on an ampicillin resistant plate and a non-resistant plate simultaneously, wherein the ampicillin resistant plate does not grow, and a pKD46 deletion strain is grown on the non-resistant plate, namely, a strain SDR1 which knocks out lacI gene and integrates sugt3 gene into the chromosome of the strain TRS1 is obtained. After liquid LB culture at 37 ℃ glycerol was stored.

Example 4 fermentation and detection of recombinant bacterium TRS1

Inoculating the recombinant strain TRS1 in LB culture medium, culturing at 37 deg.C and 220rpm under shaking for 2-4 hr, and culturing at OD₆₀₀When the concentration is between 0.8 and 1.0, transferring the mixture into M9 culture medium containing glucose to ferment for 36 hours, and synthesizing tyrosol.

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 225 nm.

The TRS1 strain was fermented in a shake flask for 36h, and the yield of tyrosol was 1.6 g/L.

Example 5 fermentation and detection of recombinant bacterium SDR1

Inoculating recombinant strain SDR1 in LB culture medium, culturing at 37 deg.C and 220rpm under shaking for 2-4 hr, and culturing at OD₆₀₀When the concentration is between 0.8 and 1.0, transferring the mixture into M9 culture medium containing glucose, and fermenting for 36h to synthesize salidroside.

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 225 nm.

The strain SDR1 is fermented for 36h in a shake flask, and the yield of salidroside is 0.7 g/L.

Sequence listing

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Claims (3)

1. A construction method of recombinant bacteria for producing salidroside is characterized by comprising the following steps:

(1) artificial total synthesis of ketodecarboxylase gene with trc promoterskdcThe sequence is shown as SEQ ID No. 1; artificial total synthetic glycosyltransferase gene with trc promotersugt2The sequence is shown as SEQ ID No. 2;

(2) will carry trc promoterskdcThe gene is integrated into the Escherichia coli SyBE-002447 by lambda-red homologous recombination technology and simultaneously knocked outfeaBGene, obtaining TRS1 strain;

(3) will carry trc promotersugt2The gene is integrated into the TRS1 strain of Escherichia coli by lambda-red homologous recombination technology, and simultaneously knocked outlacIGene, obtaining SDR1 strain;

the Escherichia coli SyBE-002447 is preserved in 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.

2. The recombinant bacterium SDR1 strain for producing salidroside constructed by the method of claim 1.

3. The method for preparing salidroside by using the recombinant salidroside-producing bacteria as claimed in claim 2, which comprises the following steps:

inoculating recombinant strain SDR1 into LB culture medium, culturing at 37 deg.C and 220rpm under shaking for 2-4 hr, and culturing at OD₆₀₀When the concentration reaches 0.8-1.0, transferring to M9 culture medium containing glucose, and fermenting for 36h to produce salidroside.