CN118064398A - Isoeugenol oxygen methyl transferase mutant and method for synthesizing hesperetin dihydrochalcone by whole cell catalysis thereof - Google Patents

Abstract

The invention discloses an isoeugenol oxygen methyl transferase mutant and a method for synthesizing hesperetin dihydrochalcone by whole cell catalysis thereof, belonging to the technical field of biology. The inventor carries out site-directed mutagenesis on IeOMT through rational design, realizes the selective methylation of 3-hydroxyphloretin in C3'-OH or C4' -OH, prepares 138mg/L hesperetin dihydrochalcone by using whole cell catalysis, and lays a foundation for synthesizing hesperetin dihydrochalcone by using enzymatic catalysis.

Description

Isoeugenol oxygen methyl transferase mutant and method for synthesizing hesperetin dihydrochalcone by whole cell catalysis thereof

Technical Field

The invention relates to an isoeugenol oxygen methyl transferase mutant and a method for synthesizing hesperetin dihydrochalcone by whole cell catalysis thereof, belonging to the technical field of biology.

Background

With the ever-changing demand for dietary sugar, research into healthy natural sweeteners has gradually led to the interest of researchers. The neohesperidin dihydrochalcone is a common natural sweetener which is commercially available at present, and the sweetness of the neohesperidin dihydrochalcone is 1800 times of that of sucrose through detection, but the sweetener has the defects of poor aftertaste like liquorice, and the like, so that the wide application of the neohesperidin dihydrochalcone is limited. The hesperidin dihydrochalcone is a novel sweetener, has sweetness equivalent to that of neohesperidin dihydrochalcone, has better and purer flavor, does not contain glycoside, and meets the requirements of healthy diet. The synthesis process of hesperidin dihydrochalcone is very few. CN110950747a discloses a method for preparing hesperetin dihydrochalcone from neohesperetin in an absolute ethanol environment through a platinum-iron-nickel three-way catalyst. Wherein the three steps of alkali liquor ring opening, hydrogenation reduction and acid hydrolysis are needed. Thus, there remains a need to explore more environmentally friendly ways to synthesize this material. However, the current method for synthesizing the hesperetin dihydrochalcone by biocatalysis is few, so that the exploration of a biosynthesis method of the substance has important significance.

In the synthetic route of plants, phloretin is used as a precursor, and 3-hydroxy phloretin can be obtained through P450 monooxygenase modification. In theory, the modification sites of 3-hydroxyphloretin by O-methyltransferase (OMTs) are typically C7, C3 'and C4'. The modification products to the C3 'and C4' sites are homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone, respectively. OMTs, however, relies on S-adenosyl-1-methionine (SAM) as a methyl donor, with strict stereoselectivity for the substrate. Mutant IeOMT _t133M of isoeugenol O-methyltransferase derived from fairy fan (Clarkia breweri) has been reported to methylate non-natural substrates naringenin, eriodictyol, luteolin, quercetin and genistein, but methylation of 3-hydroxyphloretin has not been reported. Mutants IeOMT _t133M/Y326L of isoeugenol O-methyltransferase, although altering the regioselectivity of IeOMT for flavonoid substrates, did not synthesize hesperetin dihydrochalcone (Tang, vianney et al 2020). In addition, WO2021058115Al discloses a method for biocatalytically producing dihydrochalcones. Chalcone-3-hydroxylase from sulbactam (Cosmos sulphureus) and P450 reductase from arabidopsis thaliana (Arabidopsis thaliana) are included to convert phloretin to 3-hydroxyphloretin; and methylating 3-hydroxyphloretin by a myxococcus xanthus (Myxococcus xanthus) -derived methyltransferase mutant to obtain homosanguisorba phenol dihydrochalcone or hesperetin dihydrochalcone or a mixture of both, wherein the mutant catalyzes the synthesis of hesperetin dihydrochalcone with a very low concentration (less than 45.6 mg/L) and a conversion rate of 3mM 3-hydroxyphloretin of less than 5%.

Disclosure of Invention

The invention aims to provide an isoeugenol oxymethyl transferase (IeOMT) mutant and a method for synthesizing hesperetin dihydrochalcone by whole cell catalysis.

The invention provides IeOMT mutant, which is subjected to mutation of the following sites on the basis that a starting sequence corresponds to SEQ ID NO. 2:

(1) Mutation of leucine 139 to alanine;

(2) Mutation of glutamic acid at position 165 to alanine;

(3) Mutation of glutamic acid at position 165 to arginine;

(4) Mutation of glutamic acid at position 165 to histidine;

(5) Mutation of leucine 269 to alanine;

(6) Mutating threonine at position 319 to alanine;

(7) Mutation of leucine at position 322 to alanine;

(8) Leucine 326 was mutated to histidine.

In one embodiment, the mutant is obtained by mutation on the basis of the sequence shown in SEQ ID NO. 2.

The invention also provides an expression vector containing the gene.

The invention also provides a genetic engineering bacterium for methylation of dihydrochalcone (3-hydroxy phloretin), wherein the engineering bacterium expresses isoeugenoxymethyltransferase (IeOMT) mutant derived from fairy fan (Clarkia breweri) or co-expresses 4-hydroxyphenylacetic acid 3-hydroxylase mutant (HB 03) and IeOMT mutant derived from pseudomonas aeruginosa (Pseudomonas aeruginosa).

In one embodiment, the IeOMT mutant has an amino acid sequence that is mutated on the basis of the sequence shown in SEQ ID NO. 2.

In one embodiment, HB03 has the nucleotide sequence shown in SEQ ID NO.3 and the amino acid sequence shown in SEQ ID NO. 4.

In one implementation method, the genetically engineered bacterium uses pET 28a or pET Duet1 as an expression vector.

In one embodiment, the IeOMT mutant uses pET 28a as an expression vector.

In one embodiment, the HB03 and IeOMT mutants have pET Duet1 as a co-expression vector.

In one implementation method, the genetically engineered bacterium uses escherichia coli BL 21 (DE 3) as an expression host.

The invention also provides a cell catalyst for O-methylation of 3-hydroxyphloretin, wherein the cell catalyst is a single cell catalyst or a combined cell catalyst; the single cell catalyst comprises genetically engineered bacteria co-expressing IeOMT mutants and HB03, and the combined cell catalyst comprises genetically engineered bacteria expressing IeOMT mutants and genetically engineered bacteria expressing HB 03.

In one implementation method, the genetically engineered bacteria expressing IeOMT mutant and the genetically engineered bacteria expressing HB03 are cultured in LB culture medium until OD ₆₀₀ =0.6-0.8, induced culture is performed with IPTG with a final concentration of 0.08-0.12 mM, and the whole cells are obtained by centrifugation to obtain the combined cell catalyst mixed in a ratio of 1:1 to 5:1.

The invention also provides a method for preparing the hesperetin dihydrochalcone, which comprises the step of adding the cell catalyst into a reaction system containing phloretin for reaction.

In one implementation method, the combined whole cells comprise an escherichia coli genetic engineering bacterium expressing IeOMT mutant and an escherichia coli genetic engineering bacterium expressing 4-hydroxyphenylacetic acid 3-monooxygenase.

In one implementation method, genetically engineered bacteria co-expressing IeOMT mutant and HB03 are cultured in LB medium until OD ₆₀₀ =0.6-0.8, induced with IPTG at a final concentration of 0.08-0.12 mM, and centrifuged to obtain the cell catalyst.

In one implementation method, the whole-cell catalyzed reaction system comprises 5-10% (v/v) of glycerol, 800.3-1.0% (v/v) of Tween, 500mg/L of phloretin, 1-5% (v/v) of DMSO, 4-5% (w/v) of wet thalli and 0.05-0.2 g/L of methionine.

In one embodiment, the whole cell catalyzed reaction is carried out at 30 to 40℃and 180 to 220 rpm.

The beneficial effects are that:

the invention verifies the methylation activity of IeOMT-ML on dihydrochalcone substrates (3-hydroxy phloretin) for the first time, and obtains the 3' -methylated product of the homoeriodictyol dihydrochalcone, while the wild enzyme has no activity on the 3-hydroxy phloretin.

Through rational design of IeOMT, the IeOMT mutant (ML_L139A) capable of realizing 4' -position methylation of 3-hydroxyphloretin is successfully obtained, and the selectivity to hesperetin dihydrochalcone is 100%. Under the whole-cell catalysis condition comprising coexpression HB03 and ML_L139A genetic engineering strains, the yield of hesperetin dihydrochalcone is 102.7mg/L, and the conversion rate of phloretin is 25%; under the combined whole-cell catalysis, the yield of hesperetin dihydrochalcone is 138mg/L, and the conversion rate of the hesperetin to the 3-hydroxy phloretin is 26%.

Drawings

FIG. 1 shows, from top to bottom, HPLC profiles of hesperetin dihydrochalcone standard (A), homoeriodictyol standard (B), BL21-ML and BL21-HB02 combined whole cell catalytic sample (C) and BL21-ML_139A catalytic sample (D), respectively.

FIG. 2 is a top-down LC-MS/MS diagram (A) of the hesperetin dihydrochalcone standard, respectively; LC-MS/MS plot of hesperetin dihydrochalcone in ML_139A whole cell catalytic sample (B).

FIG. 3IeOMT conversion of phloretin or 3-hydroxyphloretin by wild type and its mutants.

Fig. 4 molecular structural formulas of hesperetin dihydrochalcone and homosanguisorbal dihydrochalcone.

Detailed Description

(1) Whole cell catalytic sample treatment: dissolving the whole cell catalytic reaction sample in methanol to make the phloretin or 3-hydroxyphloretin component in the sample lower than 100mg/L, centrifuging at 12000rpm for 5min after full dissolution, collecting supernatant, and filtering with 0.22 μm filter membrane for HPLC detection.

(2) HPLC detection conditions: liquid phase separation was performed using a Waters high performance liquid chromatograph through AMETHYST C-H, 4.6x250mm column: the mobile phase was eluted isocratically at a flow rate of 1ml/min with 70% water (1% formic acid and 5% methanol) and 30% acetonitrile. The column temperature is 35 ℃, and the detection wavelength is 285nm. A single sample was run for 30min.

Example 1 catalysis of the mutant IeOMT _t133M/Y326L (ML) combination HB03 on phloretin

(1) Construction of engineering bacteria

The IeOMT _t133M/Y326L (ML) gene (SEQ ID NO. 1) was cloned between NcoI and Xhol I of vector pET 28a by a one-step cloning technique to obtain pET 28a-ML plasmid, and the HB03 gene (SEQ ID NO. 3) was cloned between BamHI and HindIII of vector pET Duet1 to obtain pET Duet1-HB03 plasmid. The plasmids which are successfully constructed are respectively transformed into escherichia coli BL21 (DE 3) to obtain corresponding engineering bacteria which are respectively named BL21-ML and BL21-HB03. The successfully constructed engineering bacteria are inoculated in 20ml of LB culture medium, cultured overnight at 37 ℃ with a shaking table at 220rpm, then transferred into 50ml of LB culture medium with an inoculum size of 1%, and added with IPTG with a final concentration of 0.1mM when the inoculum size is changed to OD ₆₀₀ = 0.6-0.8 with a shaking table at 220rpm at 37 ℃, and induced for 12h at 220rpm at 220 ℃.

(2) Combined cell catalysis

Taking the fermentation liquor obtained in the step (1), centrifuging at the temperature of 4 ℃ and the speed of 8000rpm, and respectively collecting wet thalli of BL21-ML and BL21-HB03 for combined whole cell catalysis.

The obtained BL21-ML and BL21-HB03 wet cells were mixed at a ratio of 4:1 (g/g) and resuspended as a combined cell catalyst in 100mM potassium phosphate buffer (pH=7.14), and subjected to catalytic reaction in a whole cell reaction system. The reaction system contained 10% (v/v) glycerol, 0.5% (v/v) Tween 80,0.2g/L methionine, 500mg/L phloretin, a final wet cell concentration of 5% (w/v), and a total reaction volume of 10ml. The reaction was carried out at 30℃and 200rpm, and a sample was taken after 10 hours of catalysis for HPLC and LC-MS analysis. As a result, ieOMT-T133M/Y326L, as shown in FIG. 1, can convert 3-hydroxyphloretin to homoeriodictyol dihydrochalcone. As a result of detection, the production of the eriodictyol dihydrochalcone was about 37.3mg/L in 10 hours, and the conversion was about 6.8%.

Example 2 catalysis of the Co-expression of HB03 and different IeOMT mutants on phloretin

(1) Construction of genetically engineered bacteria coexpressing IeOMT _T133M/Y326L and HB03

The gene IeOMT _T133M/Y326L (ML) (SEQ ID NO. 1) and the gene HB03 (SEQ ID NO. 3) were cloned between the BamHI and HindIII and NdeI and XholI vectors pET Duet1 by a one-step cloning technique to obtain the pET Duet1-ML-HB03 plasmid. And (3) transforming the successfully constructed plasmid into escherichia coli BL21 (DE 3) to obtain the co-expression engineering bacterium BL21-ML/HB03.

(2) Construction of IeOMT mutants

Using the above-described pET Duet1-ML-HB03 plasmid as a template, a plasmid pET Duet1-ML_L139A-HB03、pET Duet1-ML_E165A-HB03、pET Duet1-ML_E165R-HB03、pET Duet1-ML_E165H-HB03、pET Duet1-ML_W269A-HB03、pET Duet1-ML_T319A-HB03、pET Duet1-ML_L322A-HB03 expressing IeOMT mutant and pET Duet 1-ML-L326H-HB 03 were obtained by site-directed mutagenesis (the primers used are shown in Table 1). The successfully constructed plasmids were transformed into E.coli BL21 (DE 3) to obtain the corresponding engineering bacteria 139A, 165R, 165H, 269A, 319A, 322A and 326H, respectively, and the expressed mutants were named as co-expression mutants ML L139A, ML E165R, ML E165H, MLW269A, MLT319A, ML L322A and ML L326H.

TABLE 1 primers for site-directed mutagenesis

(3) Whole cell catalysis:

wet cells of the coexpression engineering bacteria and each mutant engineering bacteria were collected as a cell catalyst for whole cell catalysis according to the method described in example 1.

The cell catalyst was resuspended in 100mM potassium phosphate buffer (ph=7.14) and the catalytic reaction was performed in a whole cell reaction system. The whole cell catalytic system and conditions were the same as described in example 1, the addition of wet cells was 4-5% (w/v), and samples after 24 hours of catalysis were taken for HPLC and LC-MS analysis. As shown in FIG. 1, the methylation product of the co-expression mutant ML L139A catalytic sample was the C4' methylation product of 3-hydroxyphloretin, and the specificity for hesperetin dihydrochalcone was 100%. As shown in FIG. 2, the product was shown to be hesperetin dihydrochalcone by LC-MS analysis. The mutant was detected to produce about 102.7mg/L of hesperetin dihydrochalcone within 24 hours with a conversion rate of about 25%. The remaining mutant transformation results are shown in FIG. 3, wherein the transformation product co-expressing ML_W269A and co-expressing ML_T319A is homoeriodictyol dihydrochalcone, and the transformation product co-expressing ML_L322A is a mixture of homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone, among others.

Example 3 Combined Whole-cell catalytic Synthesis of hesperetin dihydrochalcone

Based on the pET 28a-ML plasmid in example 1, a plasmid pET 28a-ML_L139A expressing ML_L139A was constructed by using the primers described in Table 1 in the manner described in example 1, and transformed into E.coli BL21 (DE 3) to obtain the corresponding engineering bacterium BL21-ML_L139A. Then, the wet cells of BL21-HB03 and BL21-ML_L139A were collected as whole cell catalysts as described in example 1. BL21-HB03 cells (1% w/v) were first resuspended in 100mM potassium phosphate buffer (pH=7.14) (total volume of reaction system 10 ML), reacted under the above-described catalytic conditions for 3 hours to obtain a sufficient amount of 3-hydroxyphloretin, after which BL21-ML_L139A cells (4% w/v) were added to react, and a sample after catalysis for 24 hours was taken for HPLC detection. As a result, as shown in FIG. 3, the yield of hesperetin dihydrochalcone was 138mg/L, and the conversion rate of 3-hydroxyphloretin was 26%.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. An isoeugenol oxymethyl transferase mutant characterized in that on the basis of the starting sequence corresponding to SEQ ID No.2, any one of the following sites is mutated:

(1) Mutation of leucine 139 to alanine;

(2) Mutation of glutamic acid at position 165 to alanine;

(3) Mutation of glutamic acid at position 165 to arginine;

(4) Mutation of glutamic acid at position 165 to histidine;

(5) Mutation of leucine 269 to alanine;

(6) Mutating threonine at position 319 to alanine;

(7) Mutation of leucine at position 322 to alanine;

(8) Leucine 326 was mutated to histidine.

2. A gene encoding the mutant of claim 1.

3. An expression vector comprising the gene of claim 2.

4. A genetically engineered bacterium expressing the isoeugenol oxymethyl transferase mutant of claim 1.

5. An escherichia coli genetic engineering bacterium, which is characterized in that 4-hydroxyphenylacetic acid 3-monooxygenase and the isoeugenol oxymethyl transferase mutant of claim 1 are co-expressed by taking escherichia coli BL21 (DE 3) as a host and pET Duet1 as a vector; the amino acid sequence of the 4-hydroxyphenylacetic acid 3-monooxygenase is shown as SEQ ID NO. 4.

6. An escherichia coli genetically engineered bacterium, which is characterized in that escherichia coli BL21 (DE 3) is used as a host, pET28a is used as a vector, and the isoeugenol-oxymethyl transferase mutant of claim 1 is expressed.

7. A cell catalyst, characterized in that the cell catalyst is a single cell catalyst or a combination of cell catalysts; the single cell catalyst contains the escherichia coli genetically engineered bacterium of claim 5; the combined cell catalyst contains the escherichia coli genetic engineering bacteria of claim 6 and escherichia coli genetic engineering bacteria expressing 4-hydroxyphenylacetic acid 3-monooxygenase.

8. A method for preparing hesperetin dihydrochalcone, characterized in that the cell catalyst according to claim 7 is added into a whole cell catalytic reaction system containing phloretin for reaction.

9. The method of claim 8, wherein the cell catalyst is present in the reaction system in an amount of 4 to 5% w/v, and further comprising 5 to 10% v/v glycerol, 800.2 to 1.0% v/v Tween, 200 to 1000mg/L, DMSO to 5% v/v phloretin, and 0.05 to 0.2g/L methionine.

10. The mutant according to claim 1, or the genetically engineered bacterium according to any one of claims 4 to 6, for use in the synthesis of hesperetin dihydrochalcone and downstream products thereof.