Genetically engineered bacterium and method for producing icaritin through whole-cell catalysis

Abstract

The invention relates to the technical field of bioengineering, and discloses a genetically engineered bacterium and a method for producing icaritin through whole-cell catalysis. By constructing double plasmids, choline kinase, isopentenyl phosphokinase, flavonoid 3-hydroxylase, flavonol synthase, isopentenyl transferase and oxymethyltransferase are simultaneously overexpressed in escherichia coli, so that a genetically engineered bacterium is constructed, and the engineering bacterium is used as a whole-cell catalyst to prepare a whole-cell engineering bacterium. The naringenin and the 3-methyl-2-butene-1-ol are used as substrates to be converted into the icaritin in a whole-cell catalysis mode, only one-step reaction is needed, the method is simple, rapid and high in efficiency, other chemical substances do not need to be additionally added in the conversion process, the cost is reduced, pollution to the environment is relieved, and the method is suitable for industrial production. And the biological production from low-price naringenin to high-added-value icaritin is realized.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for producing icariin by genetic engineering bacteria and whole-cell catalysis. Background Art

[0002] Icaritin is a complex isopentenyl flavonoid derivative with a molecular formula of C 21 H 22 O7, with a molecular weight of 386.4, is derived from the plant of the genus Epimedium. It has a wide range of pharmacological activities and has shown great potential in the treatment of cardiovascular disease, osteoarthritis, male sexual dysfunction, inflammation, Alzheimer's disease and cancer. Icaritin soft capsules (Icaritin) have been approved by the China National Drug Administration as an anti-hepatocellular carcinoma drug for marketing.

[0003] At present, the mass production of Epimedium is mainly achieved through traditional artificial cultivation, which has problems such as long growth cycle, difficult reproduction, strict growth requirements, and low content. In addition, the chemical synthesis method faces challenges such as complex steps, harsh reaction conditions, and high pollution. In contrast, biological synthesis has the advantages of cheap raw materials, environmental friendliness, and simple process.

[0004] At present, the biosynthesis of icariin is mainly obtained by enzyme-catalyzed hydrolysis of icariin. Due to the difficulty in expressing flavonoid-3-hydroxylase, flavonol synthase, isopentenyl transferase and oxygen methyl transferase in Escherichia coli and the low conversion rate, it is currently difficult to catalyze naringenin to produce icariin in Escherichia coli. Summary of the invention

[0005] In order to solve the above technical problem of catalyzing naringenin to produce icariin in Escherichia coli, the present invention provides a genetically engineered bacterium and a method for producing icariin by whole-cell catalysis. The present invention constructs a double plasmid so that choline kinase, isopentenyl phosphate kinase, flavonoid 3-hydroxylase, flavonol synthase, isopentenyl transferase and oxygen methyl transferase are simultaneously overexpressed in Escherichia coli, thereby constructing a genetically engineered bacterium, using the engineered bacterium as a whole-cell catalyst, and converting naringenin and 3-methyl-2-butene-1-ol as substrates into icariin by whole-cell catalysis, which only requires one-step reaction, is simple and fast, and has high efficiency. No additional chemical substances need to be added during the conversion process, which reduces costs and reduces pollution to the environment, and realizes the biological production of low-priced naringenin to high-value-added icariin.

[0006] The specific technical scheme of the present invention is: On the one hand, the present invention provides a genetically engineered bacterium, which uses Escherichia coli as a base bacterium and overexpresses choline kinase, isopentenyl phosphate kinase, flavonoid 3-hydroxylase, flavonol synthase, isopentenyl transferase and oxygen methyl transferase in the base bacterium.

[0007] The invention constructs a genetically engineered bacterium by simultaneously overexpressing choline kinase, isopentenyl phosphate kinase, flavonoid 3-hydroxylase, flavonol synthase, isopentenyl transferase and oxygen methyl transferase in Escherichia coli. The engineered bacterium is used as a whole-cell catalyst to convert naringenin and 3-methyl-2-butene-1-ol as substrates into icariin through whole-cell catalysis. Only one-step reaction is required, which is simple, fast and efficient. No other chemical substances need to be added during the conversion process, which reduces costs and alleviates pollution to the environment, thereby realizing the biological production of low-cost naringenin to high-value-added icariin.

[0008] As a preferred embodiment of the above-mentioned genetically engineered bacteria, the overexpression is achieved by introducing plasmid pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS and plasmid pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 into the chassis bacteria.

[0009] The invention constructs a double plasmid to simultaneously over-express choline kinase, isopentenyl phosphate kinase, flavonoid 3-hydroxylase, flavonol synthase, isopentenyl transferase and oxygen methyl transferase in Escherichia coli, thereby constructing a genetically engineered bacterium.

[0010] As a preferred embodiment of the above-mentioned genetically engineered bacteria, the plasmid pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS is obtained by connecting the coding genes of choline kinase, isopentenyl phosphate kinase and TcF3H-(TPTP)2-CitFLS to the vector pETDuet, the TcF3H is a flavonoid 3-hydroxylase derived from the cocoa tree, and the CitFLS is a flavonol synthase derived from citrus.

[0011] As a preferred embodiment of the above-mentioned genetically engineered bacteria, the plasmid pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 is obtained by connecting the coding genes of ST-(GGGGS)2-EsPT2 and oxygen methyltransferase to the vector pACYCDuet, and the ST-(GGGGS)2-EsPT2 is a gene fragment obtained by truncating 81 amino acids starting from the N-terminal amino acid of isopentenyl transferase, mutating the 152nd threonine to asparagine, and connecting the soluble tag ST at the N-terminus with a connecting peptide (GGGGS)2.

[0012] More preferably, the ST is Trx derived from Escherichia coli.

[0013] More preferably, the Oxymethyltransferase is obtained by mutating the 135th amino acid of the wild-type Oxymethyltransferase as shown in SEQ ID NO.16 to serine and the 142nd amino acid to tyrosine.

[0014] On the other hand, the present invention provides the use of the above genetically engineered bacteria in the fermentation production of icariin.

[0015] Based on the above, the present invention also provides a method for producing icariin by whole-cell catalysis, which uses naringenin as a reaction substrate and uses the above genetically engineered bacteria to catalyze the reaction to obtain icariin.

[0016] Specifically, as a preferred embodiment of the method, after the genetically engineered bacteria are inoculated and fermented for 1 to 10 hours, IPTG is added for induction, and then substrates naringenin and 3-methyl-2-butene-1-ol are added, and the fermentation is catalytically carried out at 25 to 30° C. for 24 to 48 hours.

[0017] Compared with the prior art, the present invention has the following technical effects: The invention constructs a double plasmid so that choline kinase, isopentenyl phosphate kinase, flavonoid 3-hydroxylase, flavonol synthase, isopentenyl transferase and oxygen methyl transferase are simultaneously overexpressed in Escherichia coli, thereby constructing a genetically engineered bacterium, using the engineered bacterium as a whole-cell catalyst, and converting naringenin and 3-methyl-2-butene-1-ol as substrates into icariin through a whole-cell catalytic method, which only requires a one-step reaction, is simple and fast, and has high efficiency. No other chemical substances need to be added during the conversion process, thereby reducing costs and alleviating pollution to the environment, and realizing the biological production of low-cost naringenin to high-value-added icariin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A roadmap for whole-cell catalytic production of icariin.

[0019] Figure 2 This is the map of the recombinant plasmid pET-Scck-Atipk-F3H-(TPTP)2-FLS.

[0020] Figure 3 This is the map of the recombinant plasmid pACYC-ST-(GGGGS)2-EsPT2-MpOMT4. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0022] The enzyme involved in the embodiment of the present invention is derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) of the choline kinase Scck, from Arabidopsis thaliana ( Arabidopsis thaliana ) isopentenyl phosphate kinase Atipk, derived from Epimedium brevicornum ( Epimedium sagittatum ),Tangerine( Citrus sinensis ), Arabidopsis thaliana ( Arabidopsis thaliana ),cocoa( Theobromacacao ) from Epimedium brevicornum ( Epimedium sagittatum )、Poplar( Populusdeltoides ),Tangerine( Citrus unshiu ), Arabidopsis thaliana ( Arabidopsis thaliana )、Morning Glory( Petunia x hybrida )、Macadamia nuts( Macadamia integrifolia ) of the flavonol synthase, and the amino acid sequences are shown in SEQ ID NO.1~12 respectively.

[0023] The soluble tags involved in the embodiments of the present invention are derived from Escherichia coli ( Escherichia coli ) Trx and human ( Homo sapiens ) of Sumo, and the amino acid sequences are shown in SEQ ID NO.13~14 respectively.

[0024] The enzyme involved in the embodiment of the present invention also comes from Epimedium ( Epimedium sagittatum ) isopentenyl transferase EsPT2 from peppermint ( Menthax piperita )'s oxygen methyltransferase MpOMT4, whose amino acid sequences are shown in SEQ ID NO.15~16 respectively.

[0025] The culture medium composition involved in the embodiments of the present invention is as follows: LB liquid culture medium composition is: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, sterilized at 121°C for 20 min; LB solid culture medium composition is: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 2% agar powder, sterilized at 121°C for 20 min; TB liquid culture medium composition is: 12 g / L tryptone, 24 g / L yeast extract, 5 g / L glycerol, 2.31 g / L potassium dihydrogen phosphate, 12.5 g / L potassium dihydrogen phosphate, sterilized at 115°C for 30 min.

[0026] Example 1 Screening of F3H-Linker-FLS by Fusion Enzyme Strategy Flavonoid 3-hydroxylase and flavonol synthase successively catalyze the conversion of naringenin into dihydrokaempferol and kaempferol. In this example, four flavonoid 3-hydroxylases were first screened by adding 400 mg / L naringenin, and TcF3H from the cocoa tree was finally determined. The conversion rate results of the four flavonoid 3-hydroxylases are shown in Table 1. Subsequently, TcF3H and 6 flavonol synthases were connected using connecting peptides (GGGGS)2, (EAAAK)2 and (TPTP)2. 400 mg / L substrate naringenin was added to the 18 F3H-Linker-FLS obtained above, the kaempferol content obtained was detected, and the conversion rate of the substrate was recorded. The results are shown in Table 2. Among them: The four flavonoid 3-hydroxylases are: AtF3H from Arabidopsis thaliana, CitF3H from citrus, EsF3H from icariin, and TcF3H from theobroma cacao; The six flavonol synthases are: AtFLS from Arabidopsis thaliana, CitFLS from citrus, EsFLS from icariin, PdFLS from poplar, PhFLS from morning glory and MiFLS from macadamia.

[0027] Table 1 F3H enzyme Conversion rate (%) FWf 72.3 F3H 76.8 oeLh 69.1 T3H 80.2 Table 2 Fusion enzyme Conversion rate (%) <![CDATA[TcF3H-(GGGGS)2-AtFLS]]> 14.3 <![CDATA[TcF3H-(GGGGS)2-CitFLS]]> 19.0 <![CDATA[TcF3H-(GGGGS)2-EsFLS]]> 11.3 <![CDATA[TcF3H-(GGGGS)2-PdFLS]]> 40.0 <![CDATA[TcF3H-(GGGGS)2-PhFLS]]> 23.7 <![CDATA[TcF3H-(GGGGS)2-MiFLS]]> 33.2 <![CDATA[TcF3H-(EAAAK)2-AtFLS]]> 9.2 <![CDATA[TcF3H-(EAAAK)2-CitFLS]]> 22.6 <![CDATA[TcF3H-(EAAAK)2-EsFLS]]> 21.2 <![CDATA[TcF3H-(EAAAK)2-PdFLS]]> 11.5 <![CDATA[TcF3H-(EAAAK)2-PhFLS]]> 10.1 <![CDATA[TcF3H-(EAAAK)2-MiFLS]]> 16.2 <![CDATA[TcF3H-(TPTP)2-AtFLS]]> 11.0 <![CDATA[TcF3H-(TPTP)2-CitFLS]]> 57.3 <![CDATA[TcF3H-(TPTP)2-EsFLS]]> 13.9 <![CDATA[TcF3H-(TPTP)2-PdFLS]]> 13.8 <![CDATA[TcF3H-(TPTP)2-PhFLS]]> 7.9 <![CDATA[TcF3H-(TPTP)2-MiFLS]]> 16.4 As shown in Table 2, TcF3H-(TPTP)2-CitFLS has the best catalytic effect, with a conversion rate of 57.3%. This shows that connecting TcF3H and CitFLS through (TPTP)2 can effectively shorten the distance between the catalytic pockets, and selecting enzymes from specific sources, TcF3H and CitFLS, can effectively reduce the accumulation of intermediates and promote substrate conversion.

[0028] Example 2 Enzyme engineering to improve the catalytic efficiency of EsPT2 The transformation of this embodiment is carried out according to the following steps: (1) Isopentenyl transferase (EsPT2) can catalyze kaempferol to 8-isopentenyl kaempferol when DMAPP is in sufficient supply. Saccharomyces cerevisiae ) and the choline kinase Scck from Arabidopsis thaliana ( Arabidopsis thaliana) isopentenyl phosphate kinase Atipk, and these two enzymes can catalyze 3-methyl-2-butene-1-ol to produce DMAPP under the joint action. In this example, the isopentenyl transferase EsPT2 derived from icariin was homologously modeled and found to have a long peptide at its N-terminus. The N-terminal amino acid was then truncated to different lengths. In the case of adding 25 mM 3-methyl-2-butene-1-ol, the untruncated and truncated EsPT2 catalyzed 150 mg / L substrate kaempferol, and the conversion rate was recorded. The results are shown in Table 3. The results show that the catalytic efficiency increased by 22 times after truncating 81 amino acids starting from the N-terminal amino acid.

[0029] Table 3 EsPT2 Conversion rate (%) Scck and Atipk are not introduced 0 N-terminal not truncated 0.49 N-terminal truncation of 18 amino acids 2.09 N-terminal truncation of 26 amino acids 6.11 N-terminal truncation of 35 amino acids 4.42 N-terminal truncation of 67 amino acids 2.94 N-terminal truncation of 81 amino acids 11.06 N-terminal truncation of 90 amino acids 1.68 (2) The truncated EsPT2 was molecularly docked with the substrate kaempferol, and the sites around the pocket were selected for site-directed mutagenesis. The plasmid DNA containing the EsPT2 gene was used as a template to introduce mutations by PCR. The PCR product was treated with DpnI at 37°C for 1 hour, inactivated at 80°C for 10 minutes, and then transformed into the E. coli BL21 (DE3) recipient bacteria, spread on LB solid plates containing chloramphenicol resistance at a final concentration of 35 mg / L, and cultured at 37°C for 12 hours. Single colonies were randomly selected for sequencing analysis to obtain EsPT2 mutants, wherein the mutants were mutated from the 152th threonine of the isopentenyl transferase shown in SEQ ID NO.15 to cysteine, aspartic acid, asparagine, glycine, lysine, leucine and glutamine. The mutants were used to catalyze the conversion of kaempferol, and the catalytic results are shown in Table 4. The results showed that when the 152th threonine was mutated to asparagine, the catalytic efficiency increased by 348%.

[0030] Table 4 EsPT2 mutants Conversion rate (%) No mutation at position 152 10.6 Threonine 152 mutated to cysteine 23.4 Threonine 152 mutated to aspartic acid 0.3 Threonine 152 mutated to asparagine 36.9 Threonine 152 mutated to glycine 8.2 Threonine 152 mutated to lysine 0 Threonine 152 mutated to leucine 0 Threonine 152 mutated to glutamine 0 (3) Modeling revealed that EsPT2 has a multi-layer transmembrane structure, and it is speculated that the hydrophilicity is poor. The catalytic efficiency is further improved by connecting the soluble tag ST with a connecting peptide at the N-terminus, wherein: the Escherichia coli ( Escherichia coli ) derived soluble tag Trx, the final conversion rate reached 50.2%; connected as shown in SEQ ID NO.14 ( Homo sapiens ) with a soluble tag Sumo, and the final conversion rate reached 40.7%.

[0031] Example 3 Enzyme Engineering to Improve Catalytic Selectivity of MpOMT4 Methyltransferase MpOMT4 can catalyze the conversion of 8-isopentenylkaempferol to icariin. The mint-derived methyltransferase MpOMT4 was homologously modeled and molecularly docked with the substrate 8-isopentenylkaempferol. Sites around the pocket were selected for site-directed mutagenesis. The plasmid DNA containing the MpOMT4 gene was used as a template to introduce mutations by PCR. The PCR product was treated with DpnI at 37°C for 1h, inactivated at 80°C for 10min, and then transformed into the E. coli BL21 (DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 35mg / L chloramphenicol resistance, and cultured at 37°C for 12h. Single colonies were randomly picked for sequencing analysis to obtain MpOMT4 mutants. The results showed that the activity of amino acids 135, 137, 142, and 304 was improved after mutation to alanine or phenylalanine, but not when mutated to other amino acids. In addition, the activity of amino acid 155 to naringenin and kaempferol disappeared after mutation to alanine, but the activity to 8-isopentenylkaempferol still existed, only decreased. Then, sites 135, 142, and 155 were selected for mutation modification, and mutation points with better catalytic effect were screened for pairwise superposition mutation. Finally, it was found that the mutant obtained by mutating amino acid 135 to serine and amino acid 142 to tyrosine at the same time had the best catalytic effect.

[0032] Example 4 Construction of recombinant expression plasmid In this example, two plasmids were constructed to express icariin. The construction of the plasmids was carried out according to the following steps: (1) Construction of pET-Scck-Atipk-F3H-(TPTP)2-FLS co-expression vector This step constructs the plasmid pET-Scck-Atipk-F3H-Linker-FLS. The plasmid map is shown in Figure 2 The coding genes of Scck, Atipk and the coding gene of the TcF3H-(TPTP)2-CitFLS fragment obtained in Example 1 were amplified by PCR and connected to the vector pETDuet using a multi-fragment rapid cloning kit, so that Scck, Atipk and TcF3H-(TPTP)2-CitFLS were respectively driven by the T7 promoter to obtain the expression vector pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS.

[0033] (2) Construction of pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 co-expression vector This step constructs the plasmid pACYC-ST-(GGGGS)2-EsPT2-MpOMT4. The plasmid map is shown in Figure 3 .

[0034] According to Example 2, EsPT2 was truncated by 81 amino acids starting from the N-terminal amino acid, the 152nd threonine was mutated to asparagine, and then the Trx soluble tag ST derived from Escherichia coli was connected to the N-terminus with a connecting peptide (GGGGS) 2 to obtain ST-(GGGGS) 2-EsPT2. The coding gene of ST-(GGGGS) 2-EsPT2 and the coding gene of MpOMT4 were amplified by PCR and connected to the vector pACYCDuet using a multi-fragment rapid cloning kit, so that ST-(GGGGS) 2-EsPT2 and MpOMT4 were respectively driven by the T7 promoter to obtain the expression vector pACYC-ST-(GGGGS) 2-EsPT2-MpOMT4.

[0035] Example 5 Construction of recombinant Escherichia coli engineering bacteria The pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS plasmid constructed in Example 4 was transformed into E. coli BL21 (DE3) competent cells by heat shock method, and the product was spread on LB solid plate containing ampicillin resistance. After overnight culture, positive clones were picked and inoculated into 10 mL test tubes.

[0036] Transfer the overnight culture to 50 mL of LB liquid medium and culture until OD600 reaches 0.4-0.6. Transfer the culture to a 50 mL sterile centrifuge tube in a clean bench, centrifuge at 4°C and 5500 rpm for 5 min, and discard the supernatant. Add an appropriate amount of pre-cooled 0.1 M CaCl2 solution, gently blow the bacteria on the tube wall to suspend it, and place it on ice for 30 min. Then centrifuge at 4°C and 5500 rpm for 5 min to collect the bacteria and discard the supernatant. According to the amount of bacteria, add an appropriate amount of pre-cooled 0.1 M CaCl2, 15% glycerol solution to resuspend the cells, dispense 100 μL into each tube, add 5 μL of the pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 plasmid constructed in Example 4, transform by heat shock method, and spread the product on an LB solid plate containing ampicillin and chloramphenicol resistance. After overnight culture, pick the positive clones and inoculate them into a 10 mL test tube to obtain the icariin engineered bacteria 1, which are stored at -80°C.

[0037] Example 6 Whole-cell catalysis and yield determination Referring to Example 4, the pACYC-ST-(GGGGS)2-EsPT2-MpOMT4-2 expression vector was constructed. The only difference between this expression vector and Example 4 was that the 135th amino acid of MpOMT4 was mutated to serine and the 142nd amino acid was mutated to tyrosine. Then, referring to Example 5, pACYC-ST-(GGGGS)2-EsPT2-MpOMT4-2 and pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS were co-introduced into Escherichia coli to obtain icariin engineered bacteria 2. The wild-type icariin engineered bacteria are: referring to Example 4, constructing the pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 expression vector, the difference between the expression vector and Example 4 is that EsPT2 and MpOMT4 are not modified, and then referring to Example 5, pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 and pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS are co-introduced into Escherichia coli to obtain the wild-type icariin engineered bacteria.

[0038] (1) Shake flask fermentation The single colonies of the icariin engineered bacteria 1 and icariin engineered bacteria 2 constructed in Example 5 were inoculated into 10 mL test tubes, respectively, and ampicillin and chloramphenicol resistance were added for overnight culture. The inoculation amount was inoculated into a 250 mL shake flask containing 25 mL of fermentation medium, and ampicillin and chloramphenicol resistance were added. The shake flask fermentation was carried out at 37° C. and 200 rpm for 3 h. After induction for 10 h with the addition of IPTG at a final concentration of 0.2 mM, 400 mg / L of the substrate naringenin and 25 mM of 3-methyl-2-butene-1-ol were added, the temperature was lowered to 25° C., and catalysis was carried out for 36 h.

[0039] (2) Yield determination After the catalysis, take an appropriate amount of fermentation liquid, add 5 times the volume of methanol, mix well, centrifuge at room temperature for 5 minutes at 12000 g, take the supernatant and filter it with a 0.22 μm filter membrane for HPLC analysis. Flavonoids were detected using a Welchrom C18 HPLC Column (4.6×250 mm) and a Waters UV detector.

[0040] The HPLC detection method is as follows: phase A is pure methanol, phase B is ultrapure water containing 0.1% acetic acid, the column temperature is 35°C, the flow rate is 0.8 mL / min, the elution program is 0~5min, 45%→65% A; 5~15min, 65%→95% A; 15~25min, 95%→75% A; 25~30min, 75%→45% A, and the detection wavelength is 290nm / 360nm.

[0041] The results of the icariin production test are shown in Table 5.

[0042] Table 5 strains Icariin production (mg / L) Wild-type icariin engineered bacteria 0 Icariin engineered bacteria 1 5.1 Icariin engineered bacteria 2 15.4 As shown in Table 5, the genetically engineered bacteria containing the MpOMT mutant had the highest icariin fermentation yield. In the fermentation of this strain, choline kinase Scck can catalyze 3-methyl-2-butene-1-ol to generate isopentenyl monophosphate, which is then catalyzed by isopentenyl phosphate kinase Atipk to form DMAPP (isopentenyl donor), F3H-Linker-FLS catalyzes naringenin to generate kaempferol, and isopentenyl transferase catalyzes the substrate kaempferol and DMAPP to generate 8-isopentenyl kaempferol, which is further converted into icariin under the catalysis of MpOMT4. Figure 1 Shown is the roadmap for whole-cell catalytic production of icariin.

[0043] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0044] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that: Using Escherichia coli as the base bacteria, choline kinase, isopentenyl phosphate kinase, flavonoid 3-hydroxylase, flavonol synthase, isopentenyl transferase and oxygen methyl transferase were overexpressed in the base bacteria.

2. A genetically engineered bacterium according to claim 1, characterized in that: The overexpression is achieved by introducing plasmid pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS and plasmid pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 into the chassis bacteria.

3. A genetically engineered bacterium according to claim 2, characterized in that: The plasmid pET-Scck-Atipk-TcF3H-(TPTP)2-CitFLS is obtained by connecting the coding genes of choline kinase, isopentenyl phosphate kinase and TcF3H-(TPTP)2-CitFLS to the vector pETDuet, the TcF3H is a flavonoid 3-hydroxylase derived from the cocoa tree, and the CitFLS is a flavonol synthase derived from citrus.

4. A genetically engineered bacterium according to claim 2, characterized in that: The plasmid pACYC-ST-(GGGGS)2-EsPT2-MpOMT4 is obtained by connecting the coding genes of ST-(GGGGS)2-EsPT2 and oxygen methyltransferase to the vector pACYCDuet, and the ST-(GGGGS)2-EsPT2 is a gene fragment obtained by truncating 81 amino acids starting from the N-terminal amino acid of the isopentenyl transferase, mutating the 152nd threonine to asparagine, and connecting the soluble tag ST at the N-terminus with a connecting peptide (GGGGS)2.

5. A genetically engineered bacterium according to claim 4, characterized in that: The ST is derived from Escherichia coli.

6. A genetically engineered bacterium according to claim 4, characterized in that: The oxygen methyltransferase is obtained by mutating the 135th amino acid of the wild-type oxygen methyltransferase shown in SEQ ID NO.16 to serine and the 142nd amino acid to tyrosine.

7. Use of the genetically engineered bacteria according to any one of claims 1 to 6 in fermentation to produce icariin.

8. A method for producing icariin by whole-cell catalysis, characterized in that: Naringenin is used as a reaction substrate and icariin is obtained by catalyzing the reaction using the genetically engineered bacteria described in any one of claims 1 to 6.

9. The method according to claim 8, characterized in that: After the genetically engineered bacteria are inoculated and fermented for 1 to 10 hours, IPTG is added for induction, and then substrates naringenin and 3-methyl-2-butene-1-ol are added, and the fermentation is catalytically performed at 25 to 30° C. for 24 to 48 hours.

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