Resveratrol methylase, expression vector, expression cassette and recombinant strain

Abstract

The invention provides resveratrol methylase, an expression vector, an expression cassette and a recombinant strain. Specifically, the resveratrol synthetase is resveratrol methylase which takes S-adenosylmethionine (SAM) as a methylation reagent, and the efficient cell synthesis of the natural product pterostilbene is finally realized through the trans-dimethylation of resveratrol.

Description

Technical Field

[0001] The invention belongs to the field of synthetic biology technology, and specifically relates to a resveratrol methylase, an expression vector, an expression box and a recombinant strain. Background Art

[0002] Pterostilbene (PTS), also known as trans-3,5-dimethoxy-4-hydroxystilbene, is a substance with multiple biological activities. Its main functions include anti-tumor, cardiovascular protection, and antioxidant effects. It occupies an important position in drug synthesis and natural product applications. With the increase in public demand for cosmetics, the demand for pterostilbene is also increasing. Pterostilbene is usually a product obtained by methylation of resveratrol (RSV), a well-known stilbene derivative. However, the low bioavailability of RSV makes it ineffective under the growing demand. Therefore, in order to improve stability and bioavailability, the exploration of other highly active derivatives of RSV has been increasingly valued. Among them, PTS obtained by methylation of RSV catalyzed by trans-resveratrol di-O-methyltransferase has the most superior biological activity.

[0003] Methylation reactions are ubiquitous in nature and play a key role, such as diversifying natural products, improving the utilization rate and stability of small molecules, changing the effectiveness and cytotoxicity of natural products, regulating metabolic processes in organisms, etc. The methylation reactions involved in most biosynthesis are all catalyzed by S-adenosylmethionine (SAM)-dependent methyltransferases (MT). In recent years, it has been found that modified methyltransferases can accept complex substrates and synthesize natural products with industrial value. However, in the industrial production process with microorganisms as carriers, many reported methyltransferase activities are not enough to meet the target production requirements. Therefore, the present invention is transformed by directed evolution to improve the activity of the resveratrol methylase strain found in nature, and finally achieve the reaction activity required by industry. Summary of the invention

[0004] In order to overcome the low efficiency of natural strains of resveratrol methylase, the present invention aims to provide a resveratrol methylase that can utilize the natural methylation reagent S-adenosylmethionine to efficiently synthesize pterostilbene (such as Figure 1 shown).

[0005] Based on the existing recombinant resveratrol methylase strain, the designed resveratrol methylase (Uniprot ID: B6VJS4) mutant engineered enzyme TRdOMT was introduced to synthesize pterostilbene from resveratrol and S-adenosylmethionine.

[0006] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0007] In a first aspect of the present invention, a resveratrol methylase is provided, wherein the amino acid sequence of the resveratrol methylase is shown in SEQ ID NO:1.

[0008] In another preferred embodiment, the nucleotide sequence of the gene encoding the resveratrol methylase is shown in SEQ ID NO:2.

[0009] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the resveratrol methylase as described in the first aspect of the present invention.

[0010] In another preferred embodiment, the polynucleotide sequence is shown as SEQ ID NO:2.

[0011] In the third aspect of the present invention, an expression cassette is provided, wherein the expression cassette contains the polynucleotide according to the second aspect of the present invention.

[0012] In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide according to the second aspect of the present invention or the expression cassette according to the third aspect of the present invention.

[0013] In another preferred embodiment, the vector is a plasmid.

[0014] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector as described in the fourth aspect of the present invention, or the polynucleotide as described in the second aspect of the present invention or the expression cassette as described in the third aspect of the present invention is integrated into its genome.

[0015] In another preferred embodiment, the host cell is a prokaryotic cell.

[0016] In another preferred embodiment, the host cell is Escherichia coli or yeast.

[0017] In another preferred embodiment, the host cell contains a polynucleotide encoding pterostilbene synthase OMT.

[0018] In another preferred embodiment, the methionine repressor (MetJ) in the host cell is knocked out.

[0019] In a sixth aspect of the present invention, a method for producing pterostilbene is provided, the method comprising using the resveratrol methylase as described in the first aspect of the present invention to catalyze the methylation of resveratrol, thereby preparing pterostilbene.

[0020] In another preferred embodiment, the method comprises the steps of:

[0021] a) culturing the host cell according to the fifth aspect of the present invention, thereby producing resveratrol methylase;

[0022] b) utilizing the resveratrol methylase to catalyze the reaction between resveratrol and a methyl donor, thereby producing pterostilbene; and

[0023] c) optionally isolating pterostilbene from the culture broth.

[0024] In another preferred embodiment, the culture is shake flask culture or fermentation culture.

[0025] In another preferred embodiment, in the step b), the methyl donor is methionine or S-adenosylmethionine.

[0026] In another preferred embodiment, in the step b), the concentration of resveratrol is 2-10 g / L (preferably 5 g / L), and the concentration of methionine is 4-20 g / L (preferably 10 g / L).

[0027] In the seventh aspect of the present invention, provided is use of the resveratrol methylase according to the first aspect of the present invention, the host cell according to the fourth aspect of the present invention, or the host cell according to the fifth aspect of the present invention in producing pterostilbene.

[0028] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the reaction formula for synthesizing pterostilbene by enzymatic methylation of resveratrol. DETAILED DESCRIPTION

[0030] After in-depth research, the inventors have obtained a set of efficient biosynthetic pterostilbene system. Among them, the main design and transformation is to complete a new type of resveratrol methylase that uses S-adenosylmethionine as a methylation agent to synthesize pterostilbene from resveratrol. The new type of resveratrol methylase is designed with mutations at multiple sites based on the existing synthase to obtain the latest TRdOMT.

[0031] The obtained TRdOMT can use natural and harmless S-adenosylmethionine as a methylation agent, improve the conversion rate of resveratrol and the utilization rate of the methylation agent in genetically engineered bacteria, and ultimately significantly improve the biosynthesis efficiency of pterostilbene. Based on the above findings, the inventors have completed the present invention.

[0032] A novel resveratrol methylase using S-adenosylmethionine as a methylating agent

[0033] The invention provides a resveratrol methyltransferase TRdOMT and application thereof.

[0034] The amino acid sequence of resveratrol methylase TRdOMT is shown in SEQ ID NO: 1:

[0035] MDLANGVISAELLHAQAHVWNHIFNFIKSMSLKCAIQLGIPDIIHNHGKPMTLPELVAKLPVHPKRSQCVYRLMRILVHSGFLAAQRVQQGKEEEGYVLTDASRLLLMDDSCSIRPLVLYMLDPALTKPWHYLSAWFQNDDPTPFHRAYERSFWDYAGHEPQLNNSFNEAMASDARLPT SVLLKEGQGVFAGLNSLVDVGGGTGKVAKAIANAFPHLNCTVLDLSHVVAGLQGSKNLNYFAGDMFEAIPPADAILLKWILHDWSNEECVKILKRCREAIPSKENGGKVIIIDMIMMKNQGDYKSTETQLFFDMYMMIFAPGRERDENEWEKLFLDAGFSHYKITPILGLRSLIEVYP*;

[0036] The nucleotide sequence of its coding gene (MetS-M) is shown in SEQ ID NO: 2:

[0037] Atggatctggcgaatggcgtgatttctgcagagttgctgcatgcccaagcacatgtttggaaccacatcttcaacttcatcaagagcatgagcctgaaatgcgcaattcagctgggtattccagatattatccacaaccacggcaagccgatgaccctgccggaactggttgcaaaactgccggttcatccgaaacgttctcagtgtgtgtatcgtctgatgcgtattctcgtacacagcggtttcctggcagcacaacgtgttcagcaaggtaaagaggaagagggttatgtgctgacggatgcgtctcgtctgctgttaatggatgactcttgctctattcgtccgctggttctgtatatgctggatccggcgctgaccaaaccgtggcattatctgagcgcgtggttccagaacgatgatccgaccccgtttcatcgtgcatatgaacgtagcttctgggattatgcgggtcatgaaccgcaactgaacaacagctttaacgaggcaatggcaagcgatgcgcgtttgccgacttctgttttgctgaaagagggccagggcgtttttgcaggtctgaatagcctggtggatgttggtggtggtaccggtaaagtggcaaaagcgattgcgaacgcatttccgcatctgaattgtaccgttttagatctgtctcatgtggtggcgggtctgcagggtagcaagaacctgaactacttcgcaggtgatatgtttgaagcgattccgccggcagatgcgattctgctgaagtggattctgcacgattggagcaacgaggaatgcgtgaaaattcttaaacgttgccgtgaagccatcccgagcaaagagaacggtggcaaggtgatcatcatcgacatgatcatgatgaagaaccagggcgactacaagagcaccgaaacccagttgttcttcgacatgtatatgatgatcttcgcgccaggtcgtgaacgtgatgaaaacgagtgggaaaaactgtttctggatgcaggcttcagccactacaagatcaccccaattctgggtctgcgtagcctgatcgaagtttacccgtga

[0038] Furthermore, the amino acid sequence shown in SEQ ID NO: 1 may have one or more amino acid residues substituted, deleted, and / or added, provided that its function is not affected, and such derivative proteins with the same function also fall within the scope of protection of the present invention.

[0039] Furthermore, the nucleotide sequence shown in SEQ ID NO: 2 may have one or more nucleotide substitutions, deletions, and / or additions, provided that it does not affect its function. Such nucleotide sequences with the same function also fall within the scope of protection of the present invention.

[0040] In addition, recombinant vectors, expression cassettes, and recombinant bacteria containing the encoding gene also fall within the protection scope of the present invention.

[0041] A method for biosynthesis of pterostilbene

[0042] The present invention also provides an efficient strategy for the biosynthesis of pterostilbene. Pterostilbene is directly generated by dimethylating resveratrol, rather than the traditional natural extraction method or chemical multi-step synthesis method. And through the above-mentioned plasmid construction scheme, an engineered bacterium with high pterostilbene production is finally obtained.

[0043] Specifically, the method comprises the steps of:

[0044] (1) Transforming the TRdOMT plasmid into the engineered strain;

[0045] (2) Optionally, knocking out the methionine repressor (MetJ) of the engineered bacteria;

[0046] (3) Fermentation is carried out in a fermentation broth supplemented with resveratrol and methionine.

[0047] Preferably, the fermentation broth is supplemented with 5 g / L resveratrol and 10 g / L methionine.

[0048] TRdOMT referred to in the present invention includes resveratrol methylase and a series of resveratrol methylase mutants with equivalent methylation function that are modified, increased or decreased.

[0049] Compared with the prior art, the present invention has the following advantages and effects:

[0050] The present invention mutates the natural resveratrol methylase, so that it uses the naturally existing S-adenosylmethionine in the organism as a methylation reagent, and transports the low-cost exogenously added methionine into the cell for processing into S-adenosylmethionine as a methylation reagent, which greatly improves the production efficiency and reaction yield, and can further explore the possibility of using it as a high-yield chassis cell containing methyl natural products. Furthermore, the present invention increases the biosynthesis yield by more than 8 times for the mutation of this resveratrol methylase, showing the great potential for large-scale mass production of pterostilbene.

[0051] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. Other materials, reagents, etc. used, if not otherwise specified, are reagents and materials obtained from commercial sources.

[0052] Example 1 Synthesis of pterostilbene by shaking flask of E. coli transformed with TRdOMT

[0053] By PCR, the primers designed according to the position of the desired mutation site are combined with the template and then amplified to synthesize a vector with the target gene, and the target gene is transferred into the Escherichia coli that has been transformed into the MetJ gene after chassis cell transformation, and the plasmid is transferred into the strain in SOC medium to obtain an Escherichia coli engineered bacterium. In the following examples, the WT strain is a constructed Escherichia coli containing natural resveratrol methylase, and the TRdOMT strain is an Escherichia coli containing the resveratrol methylase mutant of the present invention (SEQ ID NO: 1).

[0054] The successfully constructed Escherichia coli engineering liquid was inoculated into 1L TB liquid culture medium at an inoculum volume of 1% (v / v), cultured at 37°C, 220rpm until OD600 = 0.8, and isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.3mM, 5g / L resveratrol, and 10g / L methionine were added, and induced expression was performed at 20°C for 24h. The content of pterostilbene was then analyzed by high performance liquid chromatography (HPLC), and the results are shown in Table 1.

[0055] Table 1. Pterostilbene content synthesized by E. coli shake flask

[0056] E. coli strains Pterostilbene(g / L) WT 1.9 TKMv 4.8

[0057] Example 2: Synthesis of pterostilbene by fermentation of Escherichia coli transforming TRdOMT

[0058] According to the inoculation amount of 2% (v / v), the Escherichia coli engineering liquid successfully cultured overnight in the shake flask was inoculated into 2L fermentation liquid (9g / L tryptone, 5g / L yeast powder, 5g / L glycerol, 12g / L potassium dihydrogen phosphate, 4g / L diamine hydrogen phosphate, pH6.9), 37°C, 200-1000rpm, 1vvm of air was ventilated to control the dissolved oxygen at 25%, and 5% ammonia was used to control the pH at 7.0. After fermentation for 3.5h, 100mL of feed medium (70% glycerol, 6g / L magnesium sulfate) was added in batches. After another 6.5h of fermentation, the temperature dropped to 20°C, IPTG with a final concentration of 0.3mM was added, and the feed medium was replaced with 500mL (70% glycerol, 6g / L magnesium sulfate, 26g / L resveratrol, 52g / L methionine) to continue fermentation for 30h. Then HPLC was used to analyze the content of pterostilbene, and the results are shown in Table 2.

[0059] Table 2. Content of pterostilbene synthesized by E. coli fermentation

[0060] E. coli strains Pterostilbene(g / L) WT 1.4 TKMv 4.3

[0061] Example 3 Synthesis of pterostilbene by directed evolution mutant strain of natural resveratrol methylase

[0062] Take 100 μL of BL21* competent cells, add 1 μL of assembled mutant strain plasmid, heat shock on ice, add 200 μL SOC medium, incubate at 37°C for 1 h, transfer to 5 mL LBK medium,

[0063] After culturing at 37°C for 16 h, the equivalent amount of strain to be added was calculated according to the obtained OD value, and the strain was transferred to 50 ml TBK medium. After 2 h at 37°C, IPTG with a final concentration of 0.3 mM was added. The strain was cultured on a shaking platform at 20°C for 24 h. The strain was collected by centrifugation and ultrasonically disrupted. 100% of the lysate was diluted to a final concentration of 1% ly, and 10 mM resveratrol, 20 mM S-adenosylmethionine, and 100 mM TrisBuffer were added. The reaction was carried out on a shaking platform at 700 rpm at 30°C and pH = 7 for 1 h. After the product was post-treated, the pterostilbene yield was observed by HPLC. The results are shown in Table 3.

[0064] Table 3. Synthetic pterostilbene content in E. coli lysate

[0065] E. coli lysate Pterostilbene(mM) WT 0.31 TKMv 8.92

[0066] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A resveratrol methylase, characterized in that The amino acid sequence of the resveratrol methylase is shown in SEQ ID NO:

1.

2. The resveratrol methylase according to claim 1, characterized in that The nucleotide sequence of the gene encoding the resveratrol methylase is shown in SEQ ID NO:

2.

3. An isolated polynucleotide, characterized in that The polynucleotide encodes the resveratrol methylase according to claim 1.

4. An expression cassette, characterized in that The expression cassette contains the polynucleotide according to claim 3.

5. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 3 or the expression cassette according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector according to claim 5, or the polynucleotide according to claim 3 or the expression cassette according to claim 4 is integrated into its genome.

7. The host cell according to claim 6, characterized in that The host cell is Escherichia coli or yeast.

8. A method for producing pterostilbene, characterized in that: The method comprises utilizing the resveratrol methylase as claimed in claim 1 to catalyze the methylation of resveratrol, thereby preparing pterostilbene.

9. The method according to claim 8, characterized in that The method comprises the steps of: a) culturing the host cell according to claim 6, thereby producing resveratrol methylase; b) utilizing the resveratrol methylase to catalyze the reaction between resveratrol and a methyl donor, thereby producing pterostilbene; and c) optionally isolating pterostilbene from the culture broth.

10. The method according to claim 8, characterized in that In the step b), the methyl donor is methionine or S-adenosylmethionine.

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