CN116904490A - Method for biosynthesis of dihydrochalcone, transgenic microorganism and construction method thereof - Google Patents
Method for biosynthesis of dihydrochalcone, transgenic microorganism and construction method thereof Download PDFInfo
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- CN116904490A CN116904490A CN202310756106.3A CN202310756106A CN116904490A CN 116904490 A CN116904490 A CN 116904490A CN 202310756106 A CN202310756106 A CN 202310756106A CN 116904490 A CN116904490 A CN 116904490A
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- dihydrochalcone
- transgenic microorganism
- biosynthesis
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- gene
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- 230000009261 transgenic effect Effects 0.000 title claims abstract description 63
- 244000005700 microbiome Species 0.000 title claims abstract description 55
- QGGZBXOADPVUPN-UHFFFAOYSA-N dihydrochalcone Chemical compound C=1C=CC=CC=1C(=O)CCC1=CC=CC=C1 QGGZBXOADPVUPN-UHFFFAOYSA-N 0.000 title claims abstract description 43
- PXLWOFBAEVGBOA-UHFFFAOYSA-N dihydrochalcone Natural products OC1C(O)C(O)C(CO)OC1C1=C(O)C=CC(C(=O)CC(O)C=2C=CC(O)=CC=2)=C1O PXLWOFBAEVGBOA-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 27
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- 238000010276 construction Methods 0.000 title abstract description 7
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- 239000005515 coenzyme Substances 0.000 claims abstract description 23
- 108010004539 Chalcone isomerase Proteins 0.000 claims abstract description 20
- 150000001875 compounds Chemical class 0.000 claims abstract description 8
- 230000008929 regeneration Effects 0.000 claims abstract description 7
- 238000011069 regeneration method Methods 0.000 claims abstract description 7
- -1 dihydrochalcone compound Chemical class 0.000 claims abstract description 4
- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 claims description 30
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- 239000007791 liquid phase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
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- 235000019139 phlorizin Nutrition 0.000 description 1
- IOUVKUPGCMBWBT-GHRYLNIYSA-N phlorizin Chemical compound O[C@@H]1[C@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 IOUVKUPGCMBWBT-GHRYLNIYSA-N 0.000 description 1
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- 150000003505 terpenes Chemical class 0.000 description 1
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- 230000009466 transformation Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
A method for biosynthesis of dihydrochalcone, a transgenic microorganism and a construction method thereof relate to the field of bioengineering and comprise the following steps: 1) Obtaining a transgenic microorganism for expressing a chalcone isomerase mutant and an enal reductase mutant, wherein the transgenic microorganism comprises a coenzyme regeneration system; 2) The transgenic microorganism is utilized to biologically catalyze the dihydroflavonoid compound into the dihydrochalcone compound. According to the method for biosynthesis of dihydrochalcone, provided by the invention, the dihydrochalcone isomerase mutant and the enal reductase mutant are expressed simultaneously, so that the dihydrochalcone can be converted into dihydrochalcone, the conversion efficiency is high, the specificity is high, and the industrialized preparation of the dihydrochalcone is realized; meanwhile, the transgenic microorganism contains a coenzyme regeneration system, so that oxidized coenzyme generates reduced coenzyme, and no additional coenzyme factor is required to be added into a whole cell system, thereby maintaining higher conversion efficiency and reducing production cost.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for biosynthesis of dihydrochalcone, a transgenic microorganism and a construction method thereof.
Background
Flavonoids (also called flavonoids) are natural products widely found in plants in nature, which are secondary metabolites of plants, and exist in the plant body in sugar-bound glycoside, carboglycosyl and free forms. Dihydrochalcones are one of the flavonoids, but are rarely distributed in nature, are called "few flavonoids", often co-exist with some flavonoids such as chalcones, flavones, flavanones, flavans, terpenes, stilbenes and the like, and exist in the form of dihydrochalcones, dihydrochalcones glycosides, dihydrochalcone derivatives and the like. At present, the compounds are obtained by separating and purifying plant species of the asteraceae, the rosaceae, the azalea, the lily and the like, and the research on the biological activity of the compounds is increasing.
The biological activity of dihydrochalcone mainly comprises the aspects of reducing blood sugar, protecting liver, resisting oxidation, resisting tumor and the like, wherein many aspects of phlorizin and phloretin are researched, and the phloretin has various important biological functions of regulating blood sugar, improving diabetes, preventing peroxidation of organisms, resisting aging, resisting inflammation and the like, and the phloretin is widely applied to skin care products and other cosmetics by virtue of the moisturizing and whitening functions, is called high-efficiency natural whitening agent, is mainly added into skin care masks, water emulsion and whitening essence, and has important utilization value. Some dihydrochalcone compounds have sweet taste and can be used as food additives, including neohesperidin dihydrochalcone (Neohesperidin Dihydrochalcone, NHDC), naringin dihydrochalcone (NaringinDihydrochalcone, NDHC) and the like, and some flavonoid compounds with bitter taste can achieve debittering effect by utilizing the characteristic of sweet taste. For example, naringin is a bitter substance that is even more bitter when citrus is processed into citrus juice, can or citrus jam. The naringin is converted into naringin dihydrochalcone, so that the bitterness of naringin can be removed, and meanwhile, the sweetness of naringin dihydrochalcone can be increased, so that the effect of having two purposes is achieved.
At present, the preparation method of the dihydrochalcone mainly adopts a chemical method, adopts the dihydroflavone as a substrate, opens a heterocycle between 1 and 2 positions under an alkaline condition to form a chalcone intermediate product, and then hydrogenates olefinic double bonds under the action of metal catalysts such as platinum, palladium and the like to finally generate the dihydrochalcone; however, the method can only be used for preparing the pure dihydrochalcone, but is difficult to realize the effect of conversion and debittering in processed foods, and meanwhile, the chemical method adopts chemical reagents which are easy to pollute the environment and easily produce byproducts, so that the method has a little potential safety hazard in the preparation of food additives, is mild in reaction condition, environment-friendly and safe, has extremely high specificity, is an ideal preparation way, and has no related report on biosynthesis of dihydrochalcones at present.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a method for biosynthesis of dihydrochalcone, which can convert the dihydrochalcone into the dihydrochalcone, and has higher conversion efficiency and strong specificity.
The second purpose of the invention is to provide a construction method of the transgenic microorganism, and construct the three-enzyme co-expressed engineering strain.
It is a further object of the present invention to provide a transgenic microorganism which can express simultaneously chalcone isomerase, enal reductase and reduced coenzyme glucose dehydrogenase and which can convert a dihydroflavone into a dihydrochalcone.
One of the purposes of the invention is realized by adopting the following technical scheme:
a method of biosynthesizing dihydrochalcones comprising the steps of:
1) Obtaining a transgenic microorganism for expressing a chalcone isomerase and an enal reductase, wherein the transgenic microorganism comprises a coenzyme regeneration system;
2) The transgenic microorganism is utilized to biologically catalyze the dihydroflavonoid compound into the dihydrochalcone compound.
Further, the amino acid sequence of the chalcone isomerase is shown in SEQ ID NO:1 is shown in the specification; the amino acid sequence of the enal reductase is shown as SEQ ID NO: 3.
Further, the transgenic microorganism comprises a gene for expressing chalcone isomerase and a gene for expressing enal reductase, wherein the nucleotide sequence of the gene for expressing chalcone isomerase is SEQ ID NO:2, wherein the nucleotide sequence of the gene for expressing the enal reductase is SEQ ID NO: 4.
Further, the coenzyme regeneration system comprises a gene encoding a glucose dehydrogenase for reducing a coenzyme. The glucose dehydrogenase is used for reducing coenzyme in the environment of existence of glucose to generate reduced coenzyme; preferably, glucose dehydrogenases include, but are not limited to, glucose dehydrogenases mentioned in enzyme classification number EC 1.1.1.47 or glucose dehydrogenases mentioned in EC 1.1.1.44.
Further, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:5 is shown in the figure; the nucleotide sequence of the coding gene of glucose dehydrogenase is shown as SEQ ID NO: shown at 6.
Further, the dihydroflavonoid compound comprises any one or more than two of naringenin, naringenin monoglucoside, naringin, hesperetin, hesperidin, neohesperidin, eriodictyol-7-O-glucoside, eriocitrin, eriodictyol, homoeriodictyol-7-O-glucoside and homoeriodictyol;
the dihydrochalcone compounds comprise one or more than two of phloretin, trilobatin, naringin dihydrochalcone, hesperetin dihydrochalcone, hesperidin dihydrochalcone, neohesperidin dihydrochalcone, 3-hydroxyphloretin monoglucoside, eriocitrin dihydrochalcone, eriodictyol dihydrochalcone, 3-methoxy phloretin monoglucoside and homoeriodictyol dihydrochalcones.
Further, in step 2), the biocatalytic reaction system comprises the following components: transgenic microbe 30-70mg/mL, dihydroflavone 500-1000 mu M and glucose 1-5mM.
The second purpose of the invention is realized by adopting the following technical scheme:
a method for constructing a transgenic microorganism used in the method for biosynthesis of dihydrochalcones, comprising the steps of:
(1) Connecting a gene for expressing chalcone isomerase into a first plasmid to obtain a first recombinant vector;
(2) Ligating a gene expressing enal reductase and a gene encoding glucose dehydrogenase for reduced coenzyme into a second plasmid to obtain a second recombinant vector;
(3) Loading the first recombinant vector and the second recombinant vector into host microorganisms, and screening to obtain transgenic microorganisms.
Further, the host microorganism includes any one or two or more of Saccharomyces cerevisiae and Escherichia coli; the first plasmid and the second plasmid respectively comprise any one or more than two of pESC-URA plasmid and pET-32a plasmid.
The third purpose of the invention is realized by adopting the following technical scheme:
a transgenic microorganism is constructed by the construction method of the transgenic microorganism.
Compared with the prior art, the invention has the beneficial effects that:
according to the method for biosynthesis of dihydrochalcone, provided by the invention, the dihydrochalcone isomerase and the enal reductase are expressed simultaneously, so that the dihydrochalcone can be converted into dihydrochalcone, the conversion efficiency is relatively high, the specificity is high, and the industrialized preparation of the dihydrochalcone is realized; at the same time, the transgenic microorganism contains a coenzyme regeneration system to oxidize the coenzyme NAD + / NADP + The reduced coenzyme NADH/NADPH is generated to participate in the reaction process of preparing dihydrochalcone, so that no additional coenzyme factor is needed to be added in a whole cell system, the higher conversion efficiency is maintained, and the production cost is reduced.
The construction method of the transgenic microorganism can construct engineering strains co-expressed by chalcone isomerase, enal reductase and reduced coenzyme glucose dehydrogenase.
The transgenic microorganism can simultaneously express chalcone isomerase, enal reductase and reduced coenzyme glucose dehydrogenase, can convert the dihydroflavone into the dihydrochalcone, and has good application prospect in debittering or sweetening of foods.
Drawings
FIG. 1 is a schematic representation of the conversion of a dihydroflavone to a dihydrochalcone by a transgenic microorganism.
FIG. 2 is a graph of a mixed standard of naringin and Naringin Dihydrochalcone (NDHC).
FIG. 3 is a graph showing the HPLC results after the transgenic microorganism reacts with naringin in example 4.
FIG. 4 is a graph showing the HPLC results after the transgenic microorganism reacts with naringin in example 5.
FIG. 5 is a graph showing the HPLC results after the reaction of the transgenic microorganism with neohesperidin in example 6.
FIG. 6 is a graph showing the HPLC results after the transgenic microorganism reacts with hesperetin in example 7.
FIG. 7 is a graph showing the HPLC results after the transgenic microorganism reacts with hesperetin monoglucoside in example 8.
FIG. 8 is a graph showing the results of HPLC after the reaction of the transgenic microorganism with eriodictyol in example 9.
FIG. 9 is a graph showing the HPLC results after the transgenic microorganism reacts with hesperidin in example 10.
FIG. 10 is a graph showing the results of HPLC after the reaction of the transgenic microorganism with eriodictyol in example 11.
FIG. 11 is a graph showing the results of HPLC after the transgenic microorganism of example 12 reacts with eriodictyol.
FIG. 12 is a graph showing the HPLC results after the transgenic microorganism reacts with eriodictyol new in example 13.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.
In the following examples, the specific conditions are specified by the conventional experimental conditions or the experimental conditions suggested by the manufacturer, and the various reagents involved in the examples are commercially available unless otherwise specified. The experimental method of molecular biology, which is not specifically described in this example, can be referred to the "guidelines for molecular cloning experiments".
Example 1
Preparation of transgenic microorganism:
1. obtaining the target gene:
synthesis of the extract from apple using total gene synthesis technologyMalus pumila) Is a chalcone isomerase mutant gene (CHI) M ) And an enal reductase mutant gene (AER M ) Synthesis of the extract from Thermoplasma acidophilumThermoplasmaacidophilum) The nucleotide sequences of the glucose dehydrogenase Genes (GDH) are respectively as shown in SEQ ID NO: 2. SEQ ID NO:4 and SEQ ID NO:6, the amino acid sequence of the expressed chalcone isomerase is shown as SEQ ID NO:1, the amino acid sequence of the enal reductase is as shown in SEQ ID NO:3, the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO: shown at 5.
2. Transgenic saccharomyces cerevisiae:
(1) The glucose dehydrogenase gene is connected to a saccharomyces cerevisiae vector frame pESC-URA, GAL1 is used as a promoter to start the expression of the glucose dehydrogenase, and the expression vector is named pESC-URA-GDH; then the chalcone isomerase gene and the enal reductase gene are simultaneously connected to a saccharomyces cerevisiae vector frame pESC-HIS, and the GAL1 and GAL10 promoters respectively start the expression of the chalcone isomerase and the enal reductase, and the expression vector is named pESC-HIS-CHI-AER.
(2) And (3) simultaneously carrying out electric shock transformation on the two expression vectors in the step (1) into saccharomyces cerevisiae BY4742, and screening through an auxotroph screening plate to obtain a positive transformant which is the transgenic saccharomyces cerevisiae and is marked as BY4742/pESC-HIS-CHI-AER/pESC-URA-GDH strain.
3. Transgenic E.coli:
(1) The glucose dehydrogenase gene is connected to an escherichia coli carrier frame pET-32a, and T7 is used as a promoter to start the expression of the glucose dehydrogenase, and the expression carrier is named pET-32a-GDH; the chalcone isomerase gene and the enal reductase gene are simultaneously connected to an escherichia coli carrier frame pET-28a, and the T7 promoter simultaneously starts the expression of the chalcone isomerase and the enal reductase, and the expression carrier is named pET-28a-CHI-AER.
(2) And (3) simultaneously carrying out heat shock conversion on the two expression vectors in the step (1) into escherichia coli BL21 (DE 3), and screening by a resistance screening plate to obtain a positive transformant which is the transgenic escherichia coli, and the positive transformant is marked as BL21 (DE 3)/pET-28 a-CHI-AER/pET-32a-GDH strain.
Example 2
Culturing and induced expression of transgenic escherichia coli:
1. configuration of the culture medium:
LB medium (100 mL): 1 g tryptone, 0.5 g yeast extract powder, 1 g sodium chloride, and sterilizing at high temperature with pure water to a volume of 100 mL.
100 x AA solution: 0.25 The gleucine+0. g lysine is prepared into 25 mL mixed aqueous solution, and the mixed aqueous solution is filtered for sterilization.
1 XYNB solution: weigh 5.36 g YNB and 11.76 g (NH) 4 ） 2 SO 4 An aqueous solution of 157.6 and mL was prepared and sterilized by filtration.
166.67 g/L galactose: 6.25/g galactose was weighed into a 37.5/mL aqueous solution and filtered for sterilization.
SD-HIS-URA liquid Medium (100 mL): 84 mL of sterilized water, 10mL of 20% glucose, 5 mL of 1 XYNB, 1mL of 100 XAA solution.
SD-HIS-URA induction Medium (50 mL): 41 mL of sterilized water, 6 mL galactose, 2.5 mL of 1 XYNB, 500. Mu.L of 100 XAA solution.
2. Induction of transgenic E.coli
BL21 (DE 3)/pET-28 a-CHI-AER/pET-32a-GDH strain of example 1 was streaked on LB plates containing Kan (100. Mu.g/mL) and Amp (100. Mu.g/mL). After being cultured overnight at 37 ℃, single colony is selected and inoculated into 5 mL LB liquid medium containing Kan and Amp, and the culture medium is cultured under shaking at 37 ℃ and 200 r/min for 12-16 h. Inoculating the overnight cultured seed solution into 100mL fresh LB liquid medium containing Kan and Amp at 37deg.C and 200 r/min shaking culture 2-3 h to OD 600 Adding IPTG with final concentration of 0.5. 0.5 mM at 0.6-0.8, and culturingThe nutrient solution is put to 37 ℃ and is subjected to shaking culture for 16 h at 200 r/min to carry out induction expression of the protein.
Example 3
Culturing and induced expression of transgenic saccharomyces cerevisiae:
the configuration of the medium of this example was the same as that of example 2;
the induction method comprises the following steps: the BY4742/pESC-HIS-CHI-AER/pESC-URA-GDH strain in example 1 was streaked on SD-HIS-URA solid medium for activation. After 2 days of inversion culture at 30℃single colonies were picked from the plates and inoculated into SD-HIS-URA liquid medium (150 mL shake flask) of 15 mL, and shake-cultured at 30℃at 200 r/min for 2 d. The seed solution in which 2 d was cultured was inoculated in 50mL fresh SD-HIS-URA induction medium (500 mL shake flask) at 1% of the inoculum size, and 3 d was shake-cultured at 30℃and 200 r/min for induction of protein expression.
Example 4
Whole-cell catalyzed production of dihydrochalcones from flavanones
1. Preparation of Whole cell enzyme solution
The transgenic E.coli of example 2 and the transgenic Saccharomyces cerevisiae of example 3 can be used for whole cell catalysis of the production of dihydrochalcone, the experiment is carried out by using the transgenic E.coli of example 2, after the induction is finished, the cells are collected by centrifugation at 5000 rpm for 5 min at 4 ℃, and after the cells are washed once by Tris-HCl buffer (20 mM, pH 7.5), an appropriate amount of Tris-HCl buffer (20 mM, pH 7.5) is added according to the wet weight of the cells to resuspend the cells, so that a cell suspension with a whole cell concentration of 100 mg/mL is obtained, and all the operations are carried out on ice or at 4 ℃.
2. Whole cell reaction
(1) Preparation of a substrate: adding propylene glycol into naringin serving as a substrate, and stirring until the naringin is fully dissolved (the naringin can be properly heated to 60 ℃) to prepare naringin reaction liquid;
(2) The reaction system shown in Table 2 was prepared using the above cell suspension as an enzyme solution:
TABLE 2
| Component (A) | Content of |
| Whole-cell enzyme solution | 50 mg/mL |
| Naringin reaction liquid | 750 μM |
| Glucose solution | 2 mM |
| Tris-HCl buffer pH 7.5 | Supplement to 1mL |
(3) The reaction system is subjected to oscillation reaction 1 d at 23 ℃; as shown in FIG. 1, in the presence of glucose solution, whole cell enzyme solution can catalyze hydrogenation and convert dihydroflavone into dihydrochalcone, and no additional coenzyme factor is needed in the reaction process. In the chemical formulas of the dihydroflavonoid compounds and the dihydrochalcone compounds shown in the figure 1, R 1 = H, OH or OMe; r is R 2 = H, OH or OMe; r is R 3 =oh, O-Glc or O-Glc-Rha (α -1,2/α -1, 6).
After the reaction, the supernatant and the precipitate were obtained by centrifugation, the precipitate was dissolved in 2 volumes of DMSO (2 mL) of the original reaction system, and after vortexing sufficiently, the cell residues were removed by centrifugation at 12,000 rpm for 1 min, and both the supernatant sample and the precipitate sample were filtered by a 0.45 μm organic filter head and subjected to HPLC detection.
3. Detection method of naringin and naringin dihydrochalcone
Quantitative analysis of naringin and naringin dihydrochalcone was performed by HPLC, and the chromatographic conditions were as follows:
high performance liquid chromatograph: agilent1100 Series
Chromatographic column: xtimateC18 XtimateC (250 mm X4.6 mm X5 μm)
A detector: VWD detector, detection wavelength 270 nm
Mobile phase ratio and elution conditions: the flow rate is 1mL/min; column temperature is 30 ℃; the sample injection amount is 10 mu L; isocratic elution system a (0.5% acetic acid water): b (acetonitrile) =66: 34, time 8 min.
4. Analysis of results
The results of the liquid phase detection of the naringin and Naringin Dihydrochalcone (NDHC) mixed standard are shown in FIG. 2, where the naringin retention time is 3.031 min and the NDHC retention time is 3.270 min. The experimental group without adding whole cell enzyme solution is used as a control group, and the detection result is shown in figure 3, so that only naringin remains in the reaction stopping solution of the control group, and no NDHC is generated; the experimental group results of the whole cell enzyme solution in the embodiment show that the conversion rate of naringin into NDHC can reach 70.73%.
Example 5
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of naringin.
As shown in FIG. 4, the transgenic microorganism can be used in the reaction of converting naringenin into phloretin, and the conversion rate is as high as 90.73%.
Example 6
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of neohesperidin.
As shown in FIG. 5, the transgenic microorganism can be used in the reaction of converting neohesperidin into neohesperidin dihydrochalcone (NHDC) with a conversion rate of 53.16%.
Example 7
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of hesperetin.
As shown in fig. 6, the transgenic microorganism can be used in a reaction for converting hesperetin into hesperetin dihydrochalcone, and the conversion rate thereof is 57.65%.
Example 8
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of hesperetin monoglucoside.
As shown in FIG. 7, the transgenic microorganism can be used in the reaction of converting hesperetin monoglucoside into hesperetin monoglucoside dihydrochalcone, and the conversion rate is 67.06%.
Example 9
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equal amount of eriodictyol.
As shown in FIG. 8, the transgenic microorganism was used in the reaction of eriodictyol to 3' -hydroxy-phloretin, and its conversion rate was 73.71%.
Example 10
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of hesperidin.
As shown in fig. 9, the transgenic microorganism can be used in the reaction of converting hesperidin into hesperidin dihydrochalcone, and the conversion rate thereof is 73.72%.
Example 11
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of homoeriodictyol.
As shown in FIG. 10, the transgenic microorganism was used in the reaction of conversion of homoeriodictyol to homoeriodictyol dihydrochalcone, and its conversion rate was 76.72%.
Example 12
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of homoeriodictyol.
As shown in FIG. 11, the transgenic microorganism was used in the reaction of converting homoeriodide into homoeriodictyol dihydrochalcone, and its conversion rate was 55.26%.
Example 13
The present embodiment differs from embodiment 4 in that: the whole cell reaction substrate naringin in example 4 was replaced with an equivalent amount of new eriodictyol.
As shown in FIG. 12, the transgenic microorganism can be used in the conversion of eriodictyol to eriodictyol dihydrochalcone with a conversion rate of 51.22%.
The result shows that the method for biosynthesizing dihydrochalcone can effectively convert the dihydrochalcone into the dihydrochalcone, has higher conversion rate on most of the dihydrochalcone compounds, has particularly outstanding conversion performance on naringin, and has important significance in the industrial production field of the dihydrochalcone and the debittering or sweetening field of foods.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (10)
1. A method of biosynthesizing dihydrochalcones, comprising the steps of:
1) Obtaining a transgenic microorganism for expressing a chalcone isomerase and an enal reductase, wherein the transgenic microorganism comprises a coenzyme regeneration system;
2) The transgenic microorganism is utilized to biologically catalyze the dihydroflavonoid compound into the dihydrochalcone compound.
2. The method of biosynthesis of dihydrochalcones according to claim 1, wherein: the amino acid sequence of the chalcone isomerase is shown in SEQ ID NO:1 is shown in the specification; the amino acid sequence of the enal reductase is shown as SEQ ID NO: 3.
3. The method for biosynthesis of dihydrochalcones according to claim 2, characterized in that: the transgenic microorganism comprises a gene for expressing chalcone isomerase and a gene for expressing enal reductase, wherein the nucleotide sequence of the gene for expressing chalcone isomerase is SEQ ID NO:2, wherein the nucleotide sequence of the gene for expressing the enal reductase is SEQ ID NO: 4.
4. The method of biosynthesis of dihydrochalcones according to claim 1, wherein: the coenzyme regeneration system comprises a gene encoding a glucose dehydrogenase for reducing a coenzyme.
5. The method for biosynthesis of dihydrochalcones according to claim 4, wherein: the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO:5 is shown in the figure; the nucleotide sequence of the coding gene of glucose dehydrogenase is shown as SEQ ID NO: shown at 6.
6. The method of biosynthesis of dihydrochalcones according to claim 1, wherein: the dihydroflavonoid compounds comprise any one or more than two of naringenin, naringenin monoglucoside, naringin, hesperetin, hesperidin, neohesperidin, eriodictyol-7-O-glucoside, eriocitrin, eriodictyol, homoeriodictyol-7-O-glucoside and homoeriodictyol;
the dihydrochalcone compounds comprise one or more than two of phloretin, trilobatin, naringin dihydrochalcone, hesperetin dihydrochalcone, hesperidin dihydrochalcone, neohesperidin dihydrochalcone, 3-hydroxyphloretin monoglucoside, eriocitrin dihydrochalcone, eriodictyol dihydrochalcone, 3-methoxy phloretin monoglucoside and homoeriodictyol dihydrochalcones.
7. The method for biosynthesis of dihydrochalcones according to claim 1, characterized in that in step 2) the biocatalytic reaction system comprises the following components: transgenic microbe 30-70mg/mL, dihydroflavone 500-1000 mu M and glucose 1-5mM.
8. A method of constructing a transgenic microorganism for use in a method of biosynthesis of dihydrochalcones according to any of the claims 1-7, characterized by comprising the steps of:
(1) Connecting a gene for expressing chalcone isomerase into a first plasmid to obtain a first recombinant vector;
(2) Ligating a gene expressing enal reductase and a gene encoding glucose dehydrogenase for reduced coenzyme into a second plasmid to obtain a second recombinant vector;
(3) Loading the first recombinant vector and the second recombinant vector into host microorganisms, and screening to obtain transgenic microorganisms.
9. The method for constructing a transgenic microorganism according to claim 8, wherein: the host microorganism comprises any one or more than two of Saccharomyces cerevisiae and Escherichia coli; the first plasmid and the second plasmid respectively comprise any one or more than two of pESC-URA plasmid and pET-32a plasmid.
10. A transgenic microorganism characterized in that: constructed by the method for constructing a transgenic microorganism according to claim 8 or 9.
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| CN117737018B (en) * | 2024-02-07 | 2024-05-03 | 广东金骏康生物技术有限公司 | Olefine aldehyde reductase mutant, rhamnosidase mutant, three-enzyme expression strain and application thereof |
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