阅读说明：本技术 单加氧酶MpdA及其编码基因mpdA以及二者在维生素E前体合成中的应用 (Monooxygenase MpdA, encoding gene mpdA thereof and application of monooxygenase MpdA and encoding gene mpdA in synthesis of vitamin E precursor ) 是由 闫新 纪俊宾 于 2021-01-19 设计创作，主要内容包括：本发明公开了单加氧酶MpdA及其编码基因mpdA以及二者在维生素E前体合成中的应用。MpdA的氨基酸序列为SEQ ID NO.2,全长413个氨基酸,其编码基因的核苷酸序列为SEQ ID NO.1,全长1242bp。MpdA是首次发现的可以催化2,3,6-三甲基苯酚转化形成维生素E前体2,3,5-三甲基氢醌的单加氧酶。含有MpdA的工程菌细胞或酶液可高效地将2,3,6-三甲基苯酚转化为2,3,5-三甲基氢醌。2,3,5-三甲基氢醌是维生素E合成的两个前体之一,2,3,6-三甲基苯酚单加氧酶MpdA与其编码基因mpdA在生物法合成维生素E前体2,3,5-三甲基氢醌中的应用潜力巨大。(The invention discloses a monooxygenase MpdA, a coding gene mpdA thereof and application of the monooxygenase MpdA and the coding gene mpdA in synthesis of a vitamin E precursor. The amino acid sequence of MpdA is SEQ ID NO.2, the total length is 413 amino acids, the nucleotide sequence of the coding gene is SEQ ID NO.1, and the total length is 1242 bp. MpdA is the first monooxygenase to be found that can catalyse the conversion of 2,3, 6-trimethylphenol to the vitamin E precursor 2,3, 5-trimethylhydroquinone. The MpdA-containing engineering bacteria cells or enzyme liquid can efficiently convert 2,3, 6-trimethylphenol into 2,3, 5-trimethylhydroquinone. 2,3, 5-trimethylhydroquinone is one of two precursors for synthesizing vitamin E, and 2,3, 6-trimethylphenol monooxygenase MpdA and an encoding gene thereof mpdA have great application potential in synthesizing the vitamin E precursor 2,3, 5-trimethylhydroquinone biologically.)

1. A2, 3, 6-trimethylphenol monooxygenase encoding gene, mpdA, comprising:

(a) has a nucleotide sequence shown as SEQ ID NO. 1; or

(b) A nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) under strict conditions and codes for a protein with 2,3, 6-trimethylphenol monooxygenase activity.

2. A2, 3, 6-trimethylphenol monooxygenase MpdA comprising:

(a) has an amino acid sequence shown as SEQ ID NO. 2;

(b) and (b) the protein which is derived from the protein (a) and has 2,3, 6-trimethylphenol monooxygenase activity, wherein the amino acid sequence in the protein (a) is substituted and/or deleted and/or added with one or more amino acids.

3. A recombinant expression vector comprising the gene mpdA encoding 2,3, 6-trimethylphenol monooxygenase according to claim 1.

4. A genetically engineered bacterium comprising the 2,3, 6-trimethylphenol monooxygenase-encoding gene, mpdA, of claim 1.

5. Use of the 2,3, 6-trimethylphenol monooxygenase encoding gene, mpdA, of claim 1 for the synthesis of the vitamin E precursor, 2,3, 5-trimethylhydroquinone.

6. Use of the 2,3, 6-trimethylphenol monooxygenase, MpdA, of claim 2 for the synthesis of the vitamin E precursor 2,3, 5-trimethylhydroquinone.

7. The use of the genetically engineered bacterium of claim 4 for the synthesis of 2,3, 5-trimethylhydroquinone, a vitamin E precursor.

8. A method for synthesizing 2,3, 5-trimethylhydroquinone as vitamin E precursor, which is characterized in that 2,3, 5-trimethylhydroquinone is biologically synthesized by catalyzing 2,3, 6-trimethylphenol with the genetically engineered bacterium as claimed in claim 4.

9. A method for synthesizing 2,3, 5-trimethylhydroquinone as a vitamin E precursor, characterized in that 2,3, 5-trimethylhydroquinone is biologically synthesized by catalyzing 2,3, 6-trimethylphenol with the 2,3, 6-trimethylphenol monooxygenase MpdA of claim 2.

Technical Field

The invention belongs to the field of high biological technology, and relates to monooxygenase MpdA, a coding gene mpdA thereof and application of the monooxygenase MpdA and the coding gene mpdA in synthesis of a vitamin E precursor.

Background

Vitamin E has multiple physiological functions of resisting oxidation, promoting sex hormone secretion, reducing cardiovascular diseases and the like, and is widely applied to the industries of food, health care products, cosmetics, clinics, medicines and the like. With the rising demand for vitamin E, the demand of people is far from being met only by extracting from leaves or seeds of plants in the nature, and more than 80% of vitamin E on the market is synthesized industrially.

Currently, the main route for the industrial synthesis of vitamin E is the condensation of 2,3, 5-trimethylhydroquinone with isophytol. The biofermentation synthesis of isophytol has been successfully commercialized in recent years, but 2,3, 5-trimethylhydroquinone is still synthesized chemically, including the 2,3, 6-trimethylphenol redox process, the isophorone process, the trimellitene redox process, and the like. Chemical synthesis is efficient but can cause environmental problems, such as the production of heavy metals and by-products required for catalytic processes. On the contrary, biosynthesis has the characteristics of environmental protection, strong specificity, low production cost and the like, and plays an increasingly important role in chemical synthesis. The biological synthesis requires specific microorganisms or enzymes, but no microorganisms or enzymes capable of specifically synthesizing 2,3, 5-trimethylhydroquinone have been known so far.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, the present invention provides 2,3, 6-trimethylphenol monooxygenase, MpdA, which converts 2,3, 6-trimethylphenol to 2,3, 5-trimethylhydroquinone, or the gene encoding mpdA, and the use of both in the synthesis of the vitamin E precursor, 2,3, 5-trimethylhydroquinone, in MpdA enzyme solution or in cells expressing MpdA.

The purpose of the invention can be realized by the following technical scheme:

the gene mpdA is derived from a strain Mycobacterium (Mycobacterium neoaurum) B5-4, the strain is preserved in China center for type culture Collection, and the strain preservation number is CCTCC NO: M2019088. The strain Mycobacterium neoaurum B5-4 is capable of degrading 2, 6-dimethylphenol and 2,3, 6-trimethylphenol.

The strategy for cloning the gene mpdA is a comparative genomic approach. A mutant strain which lost the ability to convert 2,3, 6-trimethylphenol was obtained by serial passage in LB medium (yeast powder, 5.0 g; peptone, 10.0 g; NaCl, 5.0 g; deionized water, 1L; pH 7) and was named Mycobacterium neoaurum B5-4M. By comparing the genomic sequences of the strain B5-4 and the mutant B5-4M, it was found that an about 23kb DNA fragment was lost in the mutant B5-4M; and (3) integrating bioinformatics prediction and experimental verification to obtain the target gene mpdA, wherein the coded protein is named MpdA.

The gene mpdA was ligated to vector pRESQ (van der Geize R, Hessels GI, van Gerwen R, van der Meijden P, dijkhuazine L.2002.molecular and functional characterization of kshA and kshB, encoding two compositions of 3-ketosteroid 9 alpha-hydroxyase, a class IA monoxygene, in Rhodococcus erythropolis strain SQ1.mol Microbiol 45: 1007-1018. https:// doi.org/10.1046/j.1365-2958.2002.69. X.) to obtain pMPDA 2012, which was introduced into Rhodococcus rhododendron strain Skushengi-1 (CCTCC 2017132) (Li, Zhang J.J.2002.03031. J.J.J.12. X., Zymson J.12, Q.P.P.P.J.12, J.J.S.J.S.R.R.R.R.R.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K, jiang J, Hong Q, Yan X.2017.molecular mechanism and genetic determinants of buprofazin degradation.apple Environ Microbiol 83: e00868-17.https:// doi.org/10.1128/AEM.00868-17.) to obtain the recombinant strain YL-mpdA. Cells of the Strain YL-mpdA in basal salt MSM Medium (NH)₄NO₃,1.0g；KH₂PO₄,0.5g；K₂HPO₄,1.5g；NaCl,1.0g；MgSO₄·7H₂O,0.2 g; deionized water, 1L; pH 7) is capable of converting 2,3, 6-trimethylphenol to 2,3, 5-trimethylhydroquinone and also of converting 2, 6-dimethylphenol to 2, 6-dimethylhydroquinone. Disrupting the cells of the strain YL-mpdA, wherein the disruption solution containing 2,3, 6-trimethylphenol monooxygenase MpdA converts 2,3, 6-trimethylphenol into 2,3, 5-trisMethylhydroquinone, also converts 2, 6-dimethylphenol to 2, 6-dimethylhydroquinone.

The invention relates to a 2,3, 6-trimethylphenol monooxygenase coding gene mpdA, which comprises the following components:

(a) has a nucleotide sequence shown as SEQ ID NO. 1; or

(b) A nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) under strict conditions and codes for a protein with 2,3, 6-trimethylphenol monooxygenase activity.

The invention relates to a 2,3, 6-trimethylphenol monooxygenase protein MpdA, which comprises the following components:

(a) has an amino acid sequence shown as SEQ ID NO. 2;

(b) and (b) the protein which is derived from the protein (a) and has 2,3, 6-trimethylphenol monooxygenase activity, wherein the amino acid sequence in the protein (a) is substituted and/or deleted and/or added with one or more amino acids.

The stringent conditions may be hybridization in 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65 ℃ and washing the membrane once with each of 2 XSSC, 0.1% SDS, 1 XSSC, and 0.1% SDS.

A recombinant expression vector containing the gene mpdA for coding 2,3, 6-trimethylphenol monooxygenase of the invention.

The gene engineering bacteria containing the 2,3, 6-trimethylphenol monooxygenase coding gene mpdA are disclosed.

The 2,3, 6-trimethylphenol monooxygenase coding gene mpdA disclosed by the invention is applied to synthesis of vitamin E precursor 2,3, 5-trimethylhydroquinone.

The invention relates to application of 2,3, 6-trimethylphenol monooxygenase MpdA in synthesis of vitamin E precursor 2,3, 5-trimethylhydroquinone.

The genetic engineering bacteria of the invention are applied to the synthesis of vitamin E precursor 2,3, 5-trimethylhydroquinone.

The invention relates to a method for synthesizing vitamin E precursor 2,3, 5-trimethylhydroquinone, which uses the genetic engineering bacteria to catalyze 2,3, 6-trimethylphenol to biologically synthesize the 2,3, 5-trimethylhydroquinone.

The invention relates to a method for synthesizing 2,3, 5-trimethylhydroquinone as a vitamin E precursor, which is used for biologically synthesizing 2,3, 5-trimethylhydroquinone by catalyzing 2,3, 6-trimethylphenol by using 2,3, 6-trimethylphenol monooxygenase MpdA disclosed by the invention.

Advantageous effects

1. The invention clones the 2,3, 6-trimethylphenol monooxygenase coding gene mpdA from a strain Mycobacterium neoaurum B5-4, the nucleotide sequence of the gene mpdA is SEQ ID NO.1, the full length is 1242bp, the amino acid sequence of the coded 2,3, 6-trimethylphenol monooxygenase MpdA is SEQ ID NO.2, and the full length is 413 amino acids.

2. MpdA is the first monooxygenase to be found that can catalyse the conversion of 2,3, 6-trimethylphenol to the vitamin E precursor 2,3, 5-trimethylhydroquinone. MpdA enzyme solution or cells of genetically engineered bacteria carrying mpdA can convert 2,3, 6-trimethylphenol into 2,3, 5-trimethylhydroquinone.

Drawings

FIG. 1is a schematic diagram showing the expression of the gene mpdA in the strain Rhodococcus qinshengi YL-1

FIG. 2 is a diagram of secondary mass spectrometric detection of 2,3, 5-trimethylhydroquinone produced by converting 2,3, 6-trimethylphenol with the engineered strain YL-mpdA

FIG. 3 transformation of 2,3, 6-trimethylphenol to 2,3, 5-trimethylhydroquinone by the engineered strain YL-mpdA¹H NMR chart

FIG. 4 high performance liquid chromatography detection of biological material deposit information from crude enzyme solution catalyzing 2,3, 6-trimethylphenol to 2,3, 5-trimethylhydroquinone

Mycobacterium B5-4(Mycobacterium neoaurum B5-4) is preserved in China center for type culture Collection, and the strain preservation number is CCTCC NO: M2019088. The preservation date is 2019, 1 month and 28 days, and the preservation address is Wuhan university in Wuhan, China.

Detailed Description

EXAMPLE 1 cloning of the Gene mpdA

(1) Extraction and sequencing of total DNA of bacterial genome

The strain Mycobacterium neoaurum B5-4 and the mutant strain Mycobacterium neoaurum B5-4M are respectively cultured in a liquid LB culture medium (30 ℃, 200rpm and 48h) in a large quantity, the total DNA of genomes of two strains with high purity and large fragments is extracted by adopting a CTAB method, and the two strains are respectively dissolved in deionized water and are preserved at the temperature of minus 20 ℃, and the specific method refers to the 'finely compiled molecular biology experimental manual' compiled by F.Osber and the like. The genomic DNAs of the two obtained bacteria were sequenced by sequencing company.

(2) Functional characterization of Gene mpdA

The genome of the strain Mycobacterium neoaurum B5-4 and its mutant strain is compared and analyzed, and a DNA fragment of about 23kb is lost in the mutant strain, and the fragment is analyzed in combination with genome annotation, and possible target genes are respectively connected to a vector pMV261 (Youbao, Hunan), and transformed into the mutant strain B5-4M by electric shock (transformation method reference Gordhan BG, Parish T.2001.Gene replacement using pretreated DNA. methods Mol Med 54: 77-92.). The transformant was inoculated in LB medium, cultured with shaking at 30 ℃ for 48 hours at 180rpm in a constant temperature shaker, centrifuged to collect the cells, resuspended in MSM medium, adjusted to OD600 of 1.0, added with 0.5mM 2,3, 6-trimethylphenol at 180rpm at 30 ℃ and cultured with shaking in a constant temperature shaker for 24 hours. Adding equal volume of dichloromethane to terminate the reaction and violently oscillating to extract the product, dehydrating the organic phase with anhydrous sodium sulfate, drying with nitrogen and dissolving in 100 mu L of methanol to be tested. Detecting whether the clone can be converted into 2,3, 6-trimethylphenol by using high performance liquid chromatography, wherein the liquid phase conditions are as follows: column, Kromasil 100-5C18(4.6 mm. times.250 mm. times.5 μm); mobile phase, methanol/water/phosphoric acid (volume ratio 55/44.9/0.1); flow rate, 1 ml. min^-1(ii) a Ultraviolet detectors at 275nm and 290 nm; sample size, 20. mu.l.

Storing the strain with the capability of converting 2,3, 6-trimethylphenol under the detection of high performance liquid chromatography, and naming the gene carried by the strain as mpdA, wherein the nucleotide sequence is SEQ ID NO. 1; the amino acid sequence of the protein MpdA coded by the gene mpdA is SEQ ID NO. 2.

EXAMPLE 22 Whole cell catalytic Synthesis of 3, 5-trimethylhydroquinone

(1) Construction of engineered Strain

The gene mpdA was amplified using the total DNA of strain B5-4 as a template, with the forward primer (SEQ ID NO.3) 5'-ACCGAGCTCAGATCTACTAGTATGCAATTTTCCAAAGTTGG-3' and the reverse primer (SEQ ID NO.4) 5'-ACACTGGCGGCCGTTACTAGTTCACAGCCACGGGGTATTCGG-3'. PCR amplification procedure: denaturation at 95 deg.C for 3 min; denaturation at 95 deg.C for 1.5min, annealing at 53 deg.C for 0.5min, extension at 72 deg.C for 1.5min, and performing 25 cycles; extension at 72 ℃ for 10min and cooling to room temperature. Introducing the obtained gene mpdA fragment into an SpeI enzyme digestion site of a plasmid pRESQ to construct a recombinant plasmid pMPDA; then, the recombinant plasmid was transformed into strain Rhodococcus qingshengii YL-1(CCTCC AB 2017132) (Li C, Zhang J, Wu ZG, Cao L, Yan X, Li SP.2012.Biodegr addition of bupropfezin by Rhodococcus sp.strain YL-1isolated from field soil. J agricultural Food Chem 60: 2531-2537. https:// doi.org/10.1021/jf205185n. Chen X, Ji J, Zha L, Qiu J, Dai C, Wang W, He J, Jiang J, Hong Q, Yan X.2017.molecular machinery and genetic primers of Rhodococcus strain 6317. 12. engineering strain WO 32. and J.8. engineering strain 43. 12. engineering strain 43. 12. E.J.J. 765 X.1995.tb01056.x.).

(2) Functional verification of engineering strain YL-mpdA and determination of product of 2,3, 6-trimethylphenol converted

Culturing the strain YL-mpdA in LB culture medium for 48 hours, centrifuging at 5000rpm for 10min, then suspending the bacterial cells in MSM culture medium, adjusting the bacterial concentration to OD600 ═ 1.0, adding 0.5mM of 2,3, 6-trimethylphenol, culturing at 180rpm and 30 ℃ in a constant temperature shaking table for 24-48 hours. Adding equal volume of dichloromethane to stop the reaction, violently oscillating and extracting the product, dehydrating the organic phase by anhydrous sodium sulfate, drying by nitrogen and dissolving in 100 mu L of methanol or d6-DMSO for testing. Mass spectrometry and nuclear magnetic resonance results show that the strain YL-mpdA can convert 2,3, 6-trimethylphenol into 2,3, 5-trimethylhydroquinone (figure 2 and figure 3), and can completely catalyze 0.5mM of 2,3, 6-trimethylphenol to generate corresponding 2,3, 5-trimethylhydroquinone within 48 hours, and the conversion efficiency reaches 92%.

Example enzymatic Synthesis of 32, 3, 5-trimethylhydroquinone

(1) Preparation of crude enzyme solution

Culturing the strain YL-mpdA in an LB culture medium to logarithmic phase, centrifuging at 5000rpm for 10min to collect thalli, washing twice by using a PBS buffer solution, carrying out ultrasonication in a 10ml PBS buffer solution in an ice water bath for 10-15 min (Auto Science, UH-650B ultrasonic processor, 30% intensify), centrifuging at 12000rpm for 30min, and collecting supernatant, wherein the supernatant is the prepared crude enzyme solution.

(2) Function determination of 2,3, 6-trimethylphenol catalyzed by crude enzyme liquid

The reaction system of the crude enzyme liquid catalyzing 2,3, 6-trimethylphenol is as follows: 0.5mM 2,3, 6-trimethylphenol, 0.2mM NADH,0.02mM FAD, and the crude enzyme solution to make up for 10 ml. After reacting for 2 hours at 30 ℃, adding equal volume of dichloromethane to terminate the reaction and violently oscillating to extract the product, dehydrating the organic phase by anhydrous sodium sulfate, drying by nitrogen and dissolving in 100 mu L of methanol. The peak of the substrate 2,3, 6-trimethylphenol is obviously reduced and a corresponding peak of the product 2,3, 5-trimethylhydroquinone is generated by high performance liquid chromatography (figure 4), which shows that the crude enzyme solution containing MpdA can convert 2,3, 6-trimethylphenol into the corresponding 2,3, 5-trimethylhydroquinone with the conversion efficiency of 85 percent.

Sequence listing

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