CN112011493A - Recombinant escherichia coli for producing gastrodin, construction method and application - Google Patents

Abstract

The invention discloses a recombinant escherichia coli for producing gastrodin, a construction method and application thereof, and the recombinant escherichia coli for producing gastrodin is characterized by comprising exogenous genes: 4CL gene, CCR gene, CAR gene, Sfp gene and ugt73b6^MKFAA gene. In the invention, a coenzyme A dependent reduction way is introduced for the first time in a pathway for de novo synthesis of gastrodin by using glucose as a raw material microorganism, so that an escherichia coli recombinant strain for catalytically synthesizing gastrodin is constructed, the yield of the escherichia coli recombinant strain is higher than that of recombinant escherichia coli for producing p-hydroxybenzyl alcohol or gastrodin by using glucose in the prior related patent (patent application number: 20150264006.4), and the application of the recombinant escherichia coli strain, and the gastrodin yield of the invention reaches 292 mg/L.

Description

Recombinant Escherichia coli for producing gastrodin, construction method and application

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to the application of a coenzyme A-dependent carboxylic acid reduction pathway in catalyzing the synthesis of gastrodin.

Background technique

Gastrodia elata is the stem tuber of the orchid Gastrodia elata Bl., which is a commonly used precious traditional Chinese medicine. The plant Gastrodia elata grows in sparse forests, forest clearings, forest margins, and shrub edges at an altitude of 400-3200 meters. It has been rated as a vulnerable species by the International Union for Conservation of Nature (IUCN) and is listed in the "Endangered Species of Wild Fauna and Plants". It is listed in Appendix II of the Convention on International Trade (CITES), and is also listed in China's "List of National Key Protected Wild Plants (Second Batch)" and is a Class II protected plant. Its rhizomes are used as medicine to treat dizziness, limb numbness, convulsions and convulsions in children. According to other studies, Gastrodia elata also has the functions of stimulating the nervous system, strengthening the brain, delaying aging, enhancing immunity and preventing osteoporosis. The main medicinal active ingredients of Gastrodia elata are gastrodin and its aglycone, p-hydroxybenzyl alcohol, etc. In recent years, more and more products such as medicine and food are produced with gastrodin as the main raw material. Its aglycone, p-hydroxybenzyl alcohol, is a phenolic compound with important industrial value. Synthetic precursors for various organic compounds. People pay more and more attention to gastrodin and its aglycone p-hydroxybenzyl alcohol, but so far there is no related report on the synthesis pathway of gastrodin in plants.

其苷元对羟基苄醇(4-Hydroxybenzyl alcohol，4-HBAl)具有以下特征：化学名称为p-Hydroxybenzyl alcohol，分子式为C₇H₈O₂，分子量为124.0524，CAS号为623-05-2，结构式为

Gastrodin (GAS) has the following characteristics: the chemical name is 4-(hydroxymethyl)phenylbeta-D-glucopyranoside, the molecular formula is C ₁₃ H ₁₈ O ₇ , the molecular weight is 286.1053, the CAS number is 62499-27-8, and the structural formula is

Its aglycone p-hydroxybenzyl alcohol (4-Hydroxybenzyl alcohol, 4-HBAl) has the following characteristics: the chemical name is p-Hydroxybenzyl alcohol, the molecular formula is C ₇ H ₈ O ₂ , the molecular weight is 124.0524, and the CAS number is 623-05-2 , the structural formula is

At present, the production of gastrodin is mainly through chemical synthesis and extraction of gastrodia. The chemical synthesis method requires multi-step reactions from the precursor, and there are many by-products and poor reaction specificity. In addition, the process requires the use of highly toxic bromine, red phosphorus and other substances, causing serious three-waste problems; the plant extraction method has content Too low, waste of resources, high cost, damage to the ecological environment and other defects. For example, the gastrodin content of imitation wild Gastrodia planted in Bomi County, Tibet is 0.41-2.28g/kg, and its average content is 0.88g/kg, while the Gastrodia elata of wild Gastrodia elata is 0.41-2.28g/kg. less than a quarter of that. In addition to chemical synthesis and plant extraction, microbial transformation and tissue culture production of gastrodin are also research hotspots.

Bai Yanfen et al introduced into Escherichia coli chorismate pyruvate lyase ubiC, carboxylate reductase NiCAR from Nocardia, cofactor Sfp from Bacillus subtilis, and glycosyltransferase mutant UGT73B6 from Rhodiola rosea ^FS combined with its endogenous alcohol dehydrogenase ADHs to create a pathway starting from shikimate, via p-hydroxybenzoic acid, through carboxylic acid reduction, aldehyde reduction and glycosylation synthesis pathway, the first realization of glucose as raw material microorganisms De novo synthesis of gastrodin. (BaiYF, Yin H, Bi HP, Zhuang YB, Liu T, Ma YH. De novo biosynthesis of Gastrodin in Escherichia coli [J]. Metab. Eng., 2016, 35:138-147).

The related patent that has been applied for (patent application number: 20150264006.4) utilizes glucose to produce the recombinant Escherichia coli of p-hydroxybenzyl alcohol or gastrodin and its application, wherein the output of p-hydroxybenzyl alcohol can reach 240mg/L, and the output of gastrodin can reach 265mg/ L. In order to meet the needs of industrial production, its output still needs to be further improved.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the deficiencies in the prior art, and to provide a recombinant Escherichia coli producing gastrodin that can improve gastrodin yield.

The second object of the present invention is to provide a construction method of recombinant Escherichia coli for producing gastrodin.

The third object of the present invention is to provide a use of recombinant Escherichia coli for producing gastrodin.

The technical scheme of the present invention is summarized as follows:

A recombinant Escherichia coli producing gastrodin, including exogenous genes: 4CL gene, CCR gene, CAR gene, Sfp gene and ugt73b6 ^MKFA gene.

Preferably, the 4CL gene is GenBank: X13325.1; the CCR gene is GenBank: AF332459.1; the nucleotide sequence of the CAR gene is shown in SEQ ID No: 1; the Sfp gene is from a Chinese patent Application number: 20150264006.4; Recombinant Escherichia coli using glucose to produce p-hydroxybenzyl alcohol or gastrodin and its use; the ugt73b6 ^MKFA gene is from Chinese patent application number: 201711482080.4, a glycosyltransferase mutant and its use in catalyzing gastrodin biological applications in synthesis.

The 4CL gene, CCR gene, CAR gene, Sfp gene and ugt73b6 ^MKFA gene are contained in the recombinant plasmid.

The recombinant plasmids are the first recombinant plasmid and the second recombinant plasmid, the 4CL gene and the CCR gene are contained in the first recombinant plasmid, and the CAR gene, the Sfp gene and the ugt73b6 ^MKFA gene are contained in the second recombinant plasmid.

The first recombinant plasmid is pCDFDuet-4CL-CCR; the second recombinant plasmid is pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA .

The ubiC gene was derived from Escherichia coli BL21 (DE3), GenBank: AAY88959.1.

A method for constructing recombinant Escherichia coli for gastrodin production is to introduce plasmids pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA into Escherichia coli at the same time.

The application of the above-mentioned recombinant Escherichia coli in the production of gastrodin.

The above application includes the following steps: using the above recombinant Escherichia coli, in the presence of glucose, fermenting, inducing gene expression, and obtaining a fermentation product containing gastrodin.

Advantages of the present invention:

In the present invention, a coenzyme A-dependent reduction pathway is introduced for the first time in the de novo synthesis of gastrodin by microorganisms using glucose as a raw material, and a recombinant strain of Escherichia coli for catalyzing the synthesis of gastrodin is constructed, and the yield thereof is higher than that of the existing related patents (Patent Application No. : 20150264006.4) Recombinant Escherichia coli for producing p-hydroxybenzyl alcohol or gastrodin using glucose and its application.

Description of drawings

Fig. 1 is the schematic diagram of the biosynthesis of p-hydroxybenzyl alcohol and gastrodin by recombinant Escherichia coli of the present invention, wherein: Glucose is glucose; G6P is phosphate-6-glucose; DAHP is 3-deoxy-D arabinoheptulonic acid-7 -phosphoric acid; CHA is chorismate, 4-HBAc is p-hydroxybenzoic acid, 4-HBDe is p-hydroxybenzaldehyde, 4-HBAl is p-hydroxybenzyl alcohol, 4-HBCoA is p-hydroxybenzoyl-CoA, and GAS is gastrodia elata white.

Fig. 2 is the HPLC result of the fermentation product of recombinant strain and p-hydroxybenzyl alcohol and gastrodin standard, wherein,

1 is the fermentation product of strain BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ),

2 is the fermentation product of strain BL21 (DE3, pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ),

3 is the standard product of p-hydroxybenzyl alcohol and gastrodin, peak I is the p-hydroxybenzyl alcohol peak, and peak II is the gastrodin peak.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and embodiments, and it should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

In the present invention, the carboxylic acid reductase CAR (GenBank: WP_007468889.1) is a related enzyme in the process of tyrosine synthesis of p-hydroxybenzyl alcohol. The present invention uses the genus Segniliparaceae, and other genus can also be selected.

Gene 4CL (GenBank: X13325.1) and CCR (GenBank: AF332459.1) are related genes in the formation process of cinnamyl alcohol and its derivatives, which can be derived from plants parsley and Arabidopsis thaliana, refer to the patent (patent application number: 201611179577.9 ) Recombinant Escherichia coli for producing cinnamyl alcohol and loset, construction method and application.

The gene ugt73b6 ^MKFA is a related gene in the process of synthesizing gastrodin from p-hydroxybenzyl alcohol, from a patent (patent application number: 201711482080.4) a glycosyltransferase mutant and its application in catalyzing the biosynthesis of gastrodin;

For the use of genes ubiC and Sfp, refer to the patent (patent application number: 20150264006.4) for recombinant Escherichia coli for producing p-hydroxybenzyl alcohol or gastrodin using glucose and its application.

The Sfp gene is from Chinese patent application number: 20150264006.4.

The ubiC gene was derived from Escherichia coli BL21 (DE3), GenBank: AAY88959.1.

In the present invention, it should be noted that the final recombinant Escherichia coli producing gastrodin contains ubiC, CAR, Sfp, ugt73b6 ^MKFA , 4CL and CCR genes, and can be translated into the corresponding genes during fermentation and culture in Escherichia coli protein and make the corresponding protein play its role.

In the present invention, the order of the above-mentioned genes introduced into Escherichia coli is not particularly limited as long as they can be efficiently expressed.

In the present invention, there is no special requirement for the type of the expression vector, and it can be various expression vectors commonly used in the art that can express the target gene in Escherichia coli, such as plasmids and the like. It should be understood by those skilled in the art that various methods commonly used in the art can be used for the construction of the expression vector, such as ligating the target gene into the vector after enzyme digestion, which will not be repeated here.

According to the present invention, the strains used to construct the genetically engineered bacteria can be various strains conventionally used in the art, for example, fungi, such as yeast, or bacteria, such as bacilli. According to a preferred embodiment of the present invention, the strain is Escherichia coli, and there is no special requirement for the type of Escherichia coli used to construct the Escherichia coli expression strain, and can be various Escherichia coli commonly used in the art capable of expressing the target gene. For better expression of the target gene, the E. coli is preferably BL21(DE3) and E. coli Trans-T1, more preferably E. coli strain BL21(DE3).

In the following examples, both Escherichia coli BL21(DE3) and Escherichia coli Trans-T1 are commercially available, Escherichia coli Trans-T1 is used for all gene expression in the present invention, and Escherichia coli Trans-T1 is used for all gene expression in the present invention cloning of genes.

The E. coli expression vector pCDFDuet-1 was purchased from Novagen, Cat. No. 71340. The E. coli expression vector pETDuet-1 was purchased from Novagen, Cat. No. 71146.

The test methods without specific conditions in the following examples are carried out according to conventional conditions, such as the conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions suggested by the manufacturers of the corresponding biological reagents.

Carboxylic acid reduction is one of the main rate-limiting steps in the synthesis process of gastrodin. The present invention improves the yield of aglycone p-hydroxybenzyl alcohol by introducing a coenzyme A-dependent reduction pathway, thereby further increasing the yield of gastrodin.

Example 1

Construction of Escherichia coli for Catalytic Synthesis of Gastrodin by Introducing Coenzyme A-dependent Reduction Pathway

For the construction method of the plasmid pCDFDuet-4CL-CCR, refer to the patent (patent application number: 201611179577.9), a recombinant Escherichia coli for producing cinnamyl alcohol and loset, and its construction method and application.

Plasmid pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA construction method, refer to the patent (patent application number: 20150264006.4) using glucose to produce p-hydroxybenzyl alcohol or gastrodin and its use, the plasmid pETDuet-ubiC-CAR-Sfp- The carboxylate reductase gene CAR and ugt73b6 gene from Nocardia iowensis in ugt73b6 were ligated by enzyme cleavage and replaced with the carboxylate reductase gene CAR and ugt73b6 ^MKFA gene of Segniliparaceae, respectively, to obtain the plasmid pETDuet- ubiC-CAR-Sfp-ugt73b6 ^MKFA .

The plasmids pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA were transformed into E. coli BL21(DE3) by chemical transformation. Take 100 μl BL21 competent cells on ice, add 2 μl plasmid pCDFDuet-4CL-CCR and 2 μl plasmid pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA after 10 minutes, mix gently, place on ice for 30 minutes, heat at 42°C Shock for 45 seconds, take it out and place it on ice for 2 minutes, add 900 μl LB liquid medium, 37 ° C, 150 rpm shaker for 30 minutes recovery, then spread the bacterial solution on a solution containing 25 mg/L streptomycin and 100 mg/L ampicillin. Penicillin on LB plates. The transformed strain BL21 (DE3, pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ) carrying two expression vectors at the same time was screened by streptomycin and ampicillin resistance, and the plasmid was extracted and verified by enzyme digestion. Strain BL21 was obtained (DE3, pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ).

Example 2

Fermentation culture of E. coli expression strain BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA and pCDFDuet-4CL-CCR)

The strain BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA and pCDFDuet-4CL-CCR) was cultured in 2 ml of LB liquid medium supplemented with 25 mg/L streptomycin and 100 mg/L ampicillin at 37°C After 12 hours, seed liquid was obtained.

Then, the seed liquid was transferred into 50 ml of LB liquid medium containing 25 mg/L streptomycin and 100 mg/L ampicillin according to the transfer volume (0.5 ml) of 1% by volume, and cultivated at 37 °C. When the OD ₆₀₀ was about IPTG with a final concentration of 0.1 mM was added to induce induction at 0.6 hours. After 16-18 h, the medium was replaced with M9Y liquid medium of 25 mg/L streptomycin and 100 mg/L ampicillin, and cultured at 30°C for 48 hours. Fermentation cultures of strain BL21 (DE3, pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ) were obtained.

Comparative Example 1

Fermentation culture of E. coli expression strain BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA )

The plasmid pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA was transformed into E. coli strain BL21(DE3) by chemical transformation to obtain strain BL21(DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ).

The strain BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ) was added to 2 mL of LB liquid medium with 100 mg/L ampicillin, and cultured at 37° C. for 12 hours to obtain seed liquid.

Then, the seed liquid was transferred into 50 ml of LB liquid medium with 100 mg/L ampicillin at a transfer volume of 1% by volume (0.5 ml), and cultured at 37 °C. When the OD ₆₀₀ was about 0.6, the final concentration was 0.1 mM IPTG was used for induction. After 16-18 h, the medium was replaced with 100 mg/L ampicillin M9Y liquid medium, and the culture was continued at 30°C for 48 hours. The fermentation broth of strain BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ) was obtained.

Test Example 1

Detection of hydroxybenzyl alcohol and gastrodin

(1) HPLC detection of the product: take 1 mL of each of the fermentation broths obtained in Example 2 and Comparative Example 1, centrifuge at 12,000 rpm for 10 min, and take the supernatant for HPLC analysis and detection. The analysis conditions are as follows: the instrument is an Agilent liquid chromatograph, and the measurement conditions include: C18 column (4.6×250mm); detection wavelength 224nm; mobile phase A=water (containing 0.1% formic acid by volume), B=methanol; flow rate=1mL/ min; gradient elution conditions: 0–35 min 10% volume B; injection volume 20 μL.

The HPLC detection results of the standard and fermentation broth are shown in Figure 2. in,

1 is the fermentation product of strain BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ),

2 is the fermentation product of strain BL21 (DE3, pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ),

3 is the standard product of p-hydroxybenzyl alcohol and gastrodin, peak I is the p-hydroxybenzyl alcohol peak, and peak II is the gastrodin peak.

As shown, the fermentation broths of strains BL21 (DE3, pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ) and (DE3, pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ) in 7.5 min and There is a peak at 15min, which is consistent with the peak time of p-hydroxybenzyl alcohol standard product (peak I) and gastrodin standard product (peak II). The peak II in Example 2 is significantly higher than that in Comparative Example 1.

And it was determined that the output of gastrodin in the fermentation broth of Example 2 strain BL21 (DE3, pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 ^MKFA ) was 292 mg/L, while the comparative example strain BL21 (DE3, pETDuet The yield of gastrodin in the fermentation broth of -ubiC-CAR-Sfp-ugt73b6 ^MKFA ) was 181 mg/L. In the present invention, the acetyl CoA reduction pathway is introduced for the first time in the synthesis of gastrodin, and the amount of the final product gastrodin is 1.6 times that of the comparative example. Therefore, the present invention provides a method for effectively improving the yield of gastrodin, which has certain economic value and social benefit.

sequence listing

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> Recombinant Escherichia coli for gastrodin production, construction method and application

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Claims (9)

1. A recombinant Escherichia coli for producing gastrodin, characterized by comprising exogenous genes: 4CL gene, CCR gene, CAR gene, Sfp gene and ugt73b6^MKFAA gene.

2. The recombinant Escherichia coli of claim 1, wherein the 4CL gene is GenBank: X13325.1; the CCR gene is GenBank AF 332459.1; the nucleotide sequence of the CAR gene is represented by SEQ ID No: 1 is shown in the specification; the Sfp gene is from Chinese patent application No: 20150264006.4, respectively; the ugt73b6^MKFAThe genes are from Chinese patent application numbers: 201711482080.4.

3. the recombinant Escherichia coli of claim 2, wherein said 4CL gene, CCR gene, CAR gene, Sfp gene and ugt73b6^MKFAGeneIs contained in a recombinant plasmid.

4. The recombinant Escherichia coli of claim 3, wherein said recombinant plasmids are a first recombinant plasmid and a second recombinant plasmid, said 4CL gene, CCR gene being contained in the first recombinant plasmid, said CAR gene, Sfp gene and ugt73b6^MKFAThe gene is contained in a second recombinant plasmid.

5. The recombinant E.coli of claim 4, wherein said first recombinant plasmid is pCDFDuet-4 CL-CCR; the second recombinant plasmid is pETDuet-ubiC-CAR-Sfp-ugt73b6^MKFA。

6. The recombinant Escherichia coli of claim 5, wherein said ubiC gene is derived from Escherichia coli BL21(DE3), GenBank: AAY 88959.1.

7. The method of constructing recombinant E.coli producing gastrodin according to any one of claims 1 to 6, wherein plasmids pCDFDuet-4CL-CCR and pETDuet-ubiC-CAR-Sfp-ugt73b6 b are introduced into E.coli simultaneously^MKFA。

8. Use of the recombinant escherichia coli of any one of claims 1 to 6 in production of gastrodin.

9. Use according to claim 8, characterized in that it comprises the following steps: fermenting the recombinant escherichia coli of claims 1-6 in the presence of glucose, and inducing gene expression to obtain a fermentation product containing gastrodin.

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