Preparation and application of tobacco-derived hydroxycinnamoyl-coa quinate transferase mutants

Abstract

The application discloses preparation and application of tobacco-derived hydroxycinnamoyl-CoA quinate transferase mutants and belongs to the technical field of genetic engineering and enzyme engineering. The application provides a series of tobacco-derived hydroxycinnamoyl-CoA quinate transferase mutants. The by-products of recombinant Escherichia coli of mutants P200A, P200M, P200D, A284E, T361D and P200M / A284E are reduced by 10.6%, 27.2%, 11.2%, 28.3%, 10.5% and 45.6% respectively, and the production of chlorogenic acid is increased by 33 mg / L, 62 mg / L, 32 mg / L, 63 mg / L, 35 mg / L and 88 mg / L respectively. The increase of the production of chlorogenic acid is more conducive to expanding the industrial application range.

Description

Technical Field

[0001] This invention relates to the preparation and application of tobacco-derived hydroxycinnamoyl-CoA quinic acid transferase mutants, belonging to the field of enzyme engineering technology. Background Technology

[0002] Chlorogenic acid (CGA) is a natural phenolic acid compound with the molecular formula C6H2O. 16 H 18 O9 has a relative molecular mass of 354.309. Structurally, chlorogenic acid is an ester formed by the condensation of one molecule of caffeic acid and one molecule of quinic acid. Chlorogenic acid possesses good antioxidant, antibacterial, anti-inflammatory, and neuroprotective activities, showing great application potential in the health supplement and pharmaceutical industries. Furthermore, chlorogenic acid can serve as a precursor to chicoric acid, an important compound found in echinacea. In recent years, the synthesis of chlorogenic acid has attracted increasing attention from researchers.

[0003] Currently, chlorogenic acid production mainly relies on plant extraction, but production efficiency is affected by factors such as season and low plant tissue content. Traditional chemical synthesis of chlorogenic acid involves multiple steps, resulting in low yields and unsuitability for large-scale production. Currently, chlorogenic acid synthesis has been achieved by reconstructing a microbial synthetic pathway in *E. coli* and *Saccharomyces cerevisiae* containing hydroxycinnamoyl-CoA quinic acid transferase and caffeoyl-CoA as the core. However, hydroxycinnamoyl-CoA quinic acid transferase has poor substrate specificity; while catalyzing the production of chlorogenic acid, it can also catalyze the condensation of one molecule of L-DOPA coenzyme A and one molecule of quinic acid, producing byproducts and limiting the efficient synthesis of chlorogenic acid. Therefore, screening for hydroxycinnamoyl-CoA quinic acid transferases with better substrate specificity is of great significance for modifying *E. coli* strains that produce high chlorogenic acid. Summary of the Invention

[0004] Based on the above situation, this invention utilizes genetic engineering and enzyme engineering to identify the generation of byproducts. Mutations reduce the substrate selectivity of hydroxycinnamoyl-CoA quinic acid transferase for L-DOPA-CoA, thereby reducing the generation of byproducts and creating conditions for the efficient synthesis of chlorogenic acid.

[0005] This invention provides a hydroxycinnamoyl-CoA quinic acid transferase mutant with reduced selectivity for L-DOPA-CoA substrates. This mutant is obtained by mutating one or more amino acids at positions 200, 284, or 361 of hydroxycinnamoyl-CoA quinic acid transferase derived from tobacco (Nicotiana tabacum). Compared with its parent hydroxycinnamoyl-CoA quinic acid transferase, this mutant exhibits reduced selectivity for L-DOPA-CoA.

[0006] In one embodiment, the hydroxycinnamoyl-CoA quinic acid transferase derived from tobacco (Nicotiana tabacum) has the amino acid sequence shown in SEQ ID NO.1.

[0007] In one embodiment, the mutant is obtained by mutating proline (Pro) at position 200 to alanine (Ala), and the mutant is named P200A, whose amino acid sequence is shown in SEQ ID NO.3.

[0008] In one embodiment, the mutant is a mutation of proline (Pro) at position 200 to methionine (Met), and the mutant is named P200M, whose amino acid sequence is shown in SEQ ID NO.4.

[0009] In one embodiment, the mutant is a mutation of proline (Pro) at position 200 to aspartic acid (Asp), and the mutant is named P200D, with its amino acid sequence shown in SEQ ID NO.5.

[0010] In one embodiment, the mutant is a mutation of alanine (Ala) at position 284 to glutamic acid (Glu), and the mutant is named A284E, whose amino acid sequence is shown in SEQ ID NO.6.

[0011] In one embodiment, the mutant is a mutation of threonine (Thr) at position 361 to aspartic acid (Asp), and the mutant is named T361D, with its amino acid sequence shown in SEQ ID NO.7.

[0012] In one embodiment, the mutant is a mutation of proline (Pro) at position 200 to methionine (Met) and alanine (Ala) at position 284 to glutamic acid (Glu). The mutant is named P200M / A284E and its amino acid sequence is shown in SEQ ID NO.8.

[0013] This invention provides a gene encoding the hydroxycinnamoyl-CoA quinic acid transferase mutant.

[0014] The present invention provides a plasmid carrying the gene.

[0015] In one embodiment, the plasmids include, but are not limited to, the pET series and the Duet series.

[0016] The present invention provides recombinant microbial cells expressing the tobacco-derived hydroxycinnamoyl-CoA quinic acid transferase mutant.

[0017] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0018] In one embodiment, the *Escherichia coli* includes, but is not limited to, the *E. coli* BL21(DE3) series.

[0019] In one embodiment, the recombinant Escherichia coli expresses the tobacco-derived hydroxycinnamoyl-CoA quinic acid transferase mutant using pETDuet.

[0020] The present invention also provides the application of the hydroxycinnamoyl-CoA quinic acid transferase mutant in increasing the yield of microbial chlorogenic acid.

[0021] In one embodiment, the microorganism is a microorganism capable of synthesizing chlorogenic acid.

[0022] In one embodiment, the application involves expressing the hydroxycinnamoyl-CoA quinic acid transferase mutant in Escherichia coli with a chlorogenic acid synthesis pathway.

[0023] In one embodiment, the Escherichia coli is recombinant Escherichia coli BL21(DE3)ΔtyrRΔmenI, disclosed in the paper "Efficient production of chlorogenic acid in Escherichia coli viamodular pathway and cofactor engineering".

[0024] The present invention also provides a method for producing chlorogenic acid by fermentation, wherein recombinant Escherichia coli expressing the hydroxycinnamoyl-CoA quinic acid transferase mutant is cultured in a fermentation medium to OD0.05. 600 =0.8-1.0, add IPTG to a final concentration of 0.1±0.01mM for induction.

[0025] In one embodiment, the method involves first culturing recombinant Escherichia coli in LB medium at 35–37°C for 10–12 h to obtain a seed culture, and then inoculating the seed culture into a fermentation medium for further culture.

[0026] In one implementation, fermentation lasts for at least 48 hours.

[0027] This invention provides the application of the hydroxycinnamoyl-CoA quinic acid transferase, or the gene, or the microbial cells in the production of chlorogenic acid.

[0028] The present invention also seeks protection for the use of the hydroxycinnamoyl-CoA quinic acid transferase mutant in the food, biopharmaceutical, chemical, feed, or cosmetic industries.

[0029] Beneficial Effects: This invention provides a series of mutants of tobacco-derived hydroxycinnamoyl-CoA quinic acid transferase. These mutants reduce byproduct formation and increase chlorogenic acid synthesis by decreasing substrate selectivity for L-DOPA-CoA. Under appropriate culture conditions, the byproducts of mutants P200A, P200M, P200D, A284E, T361D, and P200M / A284E were reduced by 10.6%, 27.2%, 11.2%, 28.3%, 10.5%, and 45.6%, respectively, while the chlorogenic acid yield was increased by 33 mg / L, 62 mg / L, 32 mg / L, 63 mg / L, 35 mg / L, and 88 mg / L, respectively. Attached Figure Description

[0030] Figure 1 Synthetic routes inferred for byproducts.

[0031] Figure 2 This is a liquid chromatography-mass spectrometry (LC-MS) image of wild-type WT cultured in fermentation medium after substrate feeding. Detailed Implementation

[0032] LB medium: 5g yeast extract, 10g peptone, 10g sodium chloride, add 1L deionized water for sterilization at 121℃ for 20min.

[0033] Cell culture medium (fermentation medium):

[0034] The formula consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2 g / L MgSO4·7H2O, 1 g / L sodium citrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. The 250 g / L glucose solution was sterilized separately and mixed thoroughly before inoculation.

[0035] Example 1: Identification of Byproducts Related to Chlorogenic Acid Synthesis

[0036] (1) Construction of hydroxycinnamoyl-CoA quinic acid transferase

[0037] Using hydroxycinnamoyl-CoA quinacyltransferase from tobacco (Nicotiana tabacum), as described in the paper "Efficient production of chlorogenic acid in Escherichiacoli via modular pathway and cofactor engineering," as a template, the gene fragment (nucleotide sequence shown in SEQ ID NO.2) was amplified by PCR using the designed primers HQT-F / HQT-R. After agarose gel electrophoresis, the fragment was recovered and purified. The primer sequences are shown in Table 1. The purified fragment was cloned into the vector pETDuet via homologous recombination. After amplification by E. coli JM109, DNA sequencing (Sangon, China) confirmed the correct construction of the expression plasmid (pETDuet-HQT). The successfully constructed plasmid was transformed into BL21(DE3)ΔtyrRΔmenI to obtain the recombinant strain (WT) expressing hydroxycinnamoyl-CoA quinacyltransferase.

[0038] Table 1 Primer sequences

[0039]

[0040] The PCR reaction system consisted of: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 10–50 ng of template DNA, 25 μL of 2×Phanta Max Master Mix, and double-distilled water to a final volume of 50 μL.

[0041] The PCR amplification conditions were: 95℃ pre-deformation for 3 min; followed by 30 cycles (95℃ for 15 s, 55℃ for 15 s, 72℃ for 15 s), and extension at 72℃ for 5 min.

[0042] (2) Substrate feeding to identify the formation of byproducts

[0043] The correctly sequenced recombinant plasmid was transformed into recombinant E. coli BL21(DE3)ΔtyrRΔmenI competent cells and cultured at 37℃ for 12 h. A single colony was picked and inoculated into 5 mL of LB medium, and ampicillin antibiotic was added to a final concentration of 50 μg / mL. The culture was then incubated at 37℃ and 220 rpm on a shaker for 10–12 h. The seed culture was then inoculated into 25 mL of fermentation medium with a final concentration of 50 μg / mL ampicillin antibiotic and cultured at 37℃ and 220 rpm on a shaker until OD reached [value missing]. 600When the pH reaches 0.6-0.8, add IPTG (isopropylβ-D-Thiogalactoside) to a final concentration of 0.1±0.01mM, and add levodopa and quinic acid to a final concentration of 300mg / L. Continue culturing at 30℃ and 220r / min for 12h.

[0044] High-performance liquid chromatography (HPLC): Product detection was performed using a Shimadzu LC-20A HPLC system. Chromatographic separation was performed using a ThermoFisher C18 column (4.6 mm × 250 mm, 5 μm); the column oven temperature was set to 40 °C; the injection volume was 10 μL; the mobile phases were: phase A - ultrapure water (with 0.1% trifluoroacetic acid added), and phase B - acetonitrile (with 0.1% trifluoroacetic acid added); the total flow rate was 1 mL / min; the elution mode was gradient elution: 0-10 min, phase B: 10-60%; 10-20 min, phase B: 60-80%; 20-22 min, phase B: 80-10%; 22-25 min, phase B: 10%; detector wavelength: 323 nm.

[0045] Liquid chromatography-mass spectrometry (LC-MS): Fermentation samples were dissolved in methanol and analyzed using a WatersMALDI SYNAPT Q-TOF MS platform (Milford, Massachusetts) equipped with a Waterworld Acquire UPLC BEH C18 column (2.1 mm × 50 mm) and a Watsch Acquire PDA detector (200–800 nm). MS conditions were as follows: ESI+ mode; capillary voltage, 3.5 kV; cone voltage, 30 V; source block temperature, 100 °C; desolvation gas temperature, 400 °C; desolvation gas flow rate, 700 L / h; cone gas flow rate, 50 L / h; collision energy, 6 eV; mass range, 20–2000 m / z; detector voltage, 1800 V.

[0046] By using high performance liquid chromatography and triple quadrupole liquid chromatography-mass spectrometry (LC-MS) to detect the molecular weights of all substances in the pathway, a comparative analysis was performed. Ultimately, only one combination of molecular weights matched the molecular weights in the mass spectrometer, confirming that the coenzyme A form of levodopa undergoes an esterification reaction with quinic acid under the action of HQT to generate byproducts.

[0047] Example 2: Expression of a tobacco-derived hydroxycinnamoyl-CoA quinic acid transferase mutant

[0048] (1) Construction of tobacco-derived hydroxycinnamoyl-CoA quinic acid transferase mutant

[0049] Using rapid PCR technology, primers for introducing mutations of P200A, P200M, P200D, A284E, and T361D were designed and synthesized based on the gene sequence (nucleotide sequence shown in SEQ ID NO. 2) amplified in Example 1 to perform site-directed mutagenesis on tobacco-derived hydroxycinnamoyl-CoA quinic acid transferase.

[0050] The site-directed mutagenesis primers for introducing the P200A sequence mutation are:

[0051] Forward primer: 5'-gatccgGCGacctcctccttcgaacacgttg-3',

[0052] Reverse primer: 5'-gagagtCGCcggatcacgtgcgcgc-3';

[0053] The site-directed mutagenesis primers for introducing sequences such as the P200M mutation are:

[0054] Forward primer: 5'-gatccgATGacctcctccttcgaacacgttg-3',

[0055] Reverse primer: 5'-gagagtTACcggatcacgtgcgcgc-3';

[0056] The site-directed mutagenesis primers for introducing sequences such as the P200D mutation are:

[0057] Forward primer: 5'-gatccgGATacctcctccttcgaacacgttg-3',

[0058] Reverse primer: 5'-gagagtCTAcggatcacgtgcgcgc-3';

[0059] The site-directed mutagenesis primers for introducing sequences such as the A284E mutation are:

[0060] Forward primer: 5'-cacgttGAGactgacggccgtagccg-3',

[0061] Reverse primer: 5'-gccgtcagtCTCaacgtgcagtttggtcagctg-3';

[0062] The site-directed mutagenesis primers for introducing sequences such as the T361D mutation are:

[0063] Forward primer: 5'-ggcccgGATtacttcgcgagcccgaacc-3',

[0064] Reverse primer: 5'-cgaagtaATCcgggccacggatcagc-3';

[0065] Using the constructed mutant P200M plasmid as a template, and based on the designed site-directed mutagenesis primers, rapid PCR technology was used to perform site-directed mutagenesis on the plasmid carrying the gene encoding mutant P200M, thus constructing the multimutant P200M / A284E.

[0066] The PCR reaction system consisted of: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 10–50 ng of template DNA, 25 μL of 2×Phanta Max Master Mix, and double-distilled water to a final volume of 50 μL.

[0067] The PCR amplification conditions were: 95℃ pre-deformation for 3 min; followed by 30 cycles (95℃ for 15 s, 55℃ for 15 s, 72℃ for 15 s), and extension at 72℃ for 5 min.

[0068] After the PCR products were verified to be correct, they were digested with DpnI and transformed into competent E. coli JM109 cells. The competent cells were cultured overnight in LB solid medium (containing 50 mg / L ampicillin). After culturing the cells in LB liquid medium (containing 50 mg / L ampicillin), clones were picked and cultured in LB liquid medium (containing 50 mg / L ampicillin). Plasmids were then extracted. All mutant plasmids were correctly sequenced and named P200A, P200M, P200D, A284E, T361D, and P200M / A284E, respectively.

[0069] (2) Expression of mutant enzymes

[0070] The mutant plasmids constructed in step (1) were transformed into *E. coli* BL21(DE3)ΔtyrRΔmenI for expression. Single colonies were picked and inoculated into test tubes containing 5 mL of LB medium, and cultured at 37°C and 220 rpm for 10–12 h to obtain seed culture. The seed culture was then inoculated at a 2% inoculation rate into 50 mL of ampicillin antibiotic fermentation medium at a final concentration of 50 μg / mL, and cultured at 37°C and 220 rpm on a shaker until OD500 reached. 600 When the pH reaches between 0.6 and 0.8, add IPTG (isopropylβ-D-Thiogalactoside) to a final concentration of 0.1 ± 0.01 mM and continue culturing for 12 h.

[0071] Example 3: Comparison of shake-flask levels of hydroxycinnamoyl-CoA quinic acid transferase mutants

[0072] The plasmids P200A, P200M, P200D, A284E, T361D, and P200M / T361D that were correctly sequenced in Example 2 were introduced into recombinant Escherichia coli BL21(DE3)ΔtyrRΔmenI (published in the paper "Efficient production of chlorogenic acid in Escherichia coli via modular pathway and cofactor engineering") to produce chlorogenic acid-producing Escherichia coli expressing a hydroxycinnamoyl-CoA quinic acid transferase mutant. The recombinant strain was cultured according to the method in Example 2, and the amount of byproducts and chlorogenic acid produced in the fermentation broth after 48 hours of culture was detected.

[0073] The results showed that the peak area of ​​byproducts of each mutant was lower than that of the wild type (WT). The byproducts of recombinant Escherichia coli expressing P200A, P200M, P200D, A284E, T361D, and P200M / A284E were reduced by 10.6%, 27.2%, 11.2%, 28.3%, 10.5%, and 45.6%, respectively, while the yield of chlorogenic acid was increased by 33 mg / L, 62 mg / L, 32 mg / L, 63 mg / L, 35 mg / L, and 88 mg / L, respectively.

[0074] Table 2. Peak areas of byproducts and chlorogenic acid yield of wild-type hydroxycinnamoyl-CoA quinolone transferase and mutant enzyme.

[0075]

[0076] Comparative Example 1:

[0077] The specific implementation method is the same as in Example 2, except that mutants with the mutations shown in Table 3 were also constructed and fermented according to the method in Example 3. The results showed that the peak area of ​​the byproducts generated by these mutants was greater than that of wild-type hydroxycinnamoyl-CoA quinic acid transferase (peak area 840915), and the yield of chlorogenic acid was lower than that of WT (554 mg / L). The effect was far inferior to that of the mutants constructed in Example 2.

[0078] Table 3. Peak area of ​​byproducts and chlorogenic acid yield of different mutant enzymes.

[0079]

[0080]

[0081] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A hydroxycinnamoyl-CoA quinic acid transferase mutant, characterized in that, Compared to the amino acid sequence shown in SEQ ID NO.1, only one mutation selected from (a) to (f) exists: (a) Mutate the 200th proline to alanine; (b) Mutate proline at position 200 to methionine; (c) Mutate proline at position 200 to aspartic acid; (d) Mutate alanine at position 284 to glutamic acid; (e) Mutate threonine at position 361 to aspartic acid; (f) Mutate proline at position 200 to methionine and alanine at position 284 to glutamic acid.

2. The gene encoding the hydroxycinnamoyl-CoA quinic acid transferase mutant of claim 1.

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

4. A microbial cell expressing the mutant of claim 1, or containing the gene of claim 2.

5. A recombinant Escherichia coli, characterized in that, The *E. coli* BL21(DE3) Δ expressing the hydroxycinnamoyl-CoA quinic acid transferase mutant of claim 1 tyrR Δ menI .

6. The application of the hydroxycinnamoyl-CoA quinic acid transferase mutant according to claim 1 in increasing microbial chlorogenic acid production, characterized in that, The microorganism in question is Escherichia coli.

7. A method for producing chlorogenic acid by fermentation, characterized in that, Recombinant Escherichia coli expressing the hydroxycinnamoyl-CoA quinic acid transferase mutant of claim 1 was cultured in fermentation medium to OD. 600 =0.8-1.0, add IPTG to a final concentration of 0.1±0.01 mM for induction.

8. The method according to claim 7, characterized in that, The fermentation medium contains glucose, glycerol, (NH4)2SO4, K2HPO4·3H2O, and KH2PO4. 4、 MgSO4·7H2O, sodium citrate, vitamin B1 and yeast extract.

9. The application of the hydroxycinnamoyl-CoA quinic acid transferase mutant of claim 1, or the gene of claim 2, or the microbial cell of claim 4, or the recombinant Escherichia coli of claim 5 in the production of chlorogenic acid, characterized in that, The microorganism in question is Escherichia coli.

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