A phenylalanine hydroxylase mutant and its application in the synthesis of tryptophan derivatives

Abstract

This invention belongs to the fields of biocatalysis and biopharmaceuticals, specifically relating to a phenylalanine hydroxylase mutant and its application in the synthesis of tryptophan derivatives. This invention utilizes genome editing to construct a high-L-tryptophan-producing *E. coli* strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL), and uses this strain as a chassis cell to overexpress the following enzymes in different combinations: a phenylalanine hydroxylase mutant (XsP4H_m), a dehydratase (CvPcd), a reductase (EcFolM), a decarboxylase (CkDdc), an acetyltransferase (SgAanat), and a methyltransferase (HsAsmt). Tryptophan derivatives such as 5-HTP, 5-HT, or melatonin are synthesized using biofermentation methods, achieving highly efficient biosynthesis of tryptophan derivatives at room temperature and atmospheric pressure.

Description

Technical Field

[0001] This invention belongs to the fields of biocatalysis and biopharmaceuticals, specifically relating to a phenylalanine hydroxylase mutant and its application in the synthesis of tryptophan derivatives. Background Technology

[0002] Tryptophan is one of the essential amino acids for the human body and has important physiological functions. 5-Hydroxytryptophan (5-HTP) is a tryptophan derivative that does not participate in protein synthesis. It is an important precursor to the neurotransmitter serotonin and the amine hormone melatonin, and plays a crucial regulatory role in disorders and diseases such as depression, insomnia, Parkinson's disease, and Alzheimer's disease. Serotonin (5-HT), also known as serotonin, is an important signaling molecule in the human central and peripheral nervous systems. It helps regulate the normal function of the gastrointestinal tract, cardiovascular system, and brain, and is also associated with the etiology of various diseases, including depression, anxiety, hypertension, and irritable bowel syndrome. Melatonin (N-acetyl-5-methoxytryptamine) is a biogenic amine that has gradually become well-known for its anti-inflammatory, antioxidant, and autophagy-promoting properties. In plants, melatonin levels affect their response to biotic and abiotic stresses; in animals, it can regulate sleep, body temperature, and increase dairy production; in humans, it can protect lymphocytes from DNA damage caused by ionizing radiation, regulate circadian rhythms, and protect the skin.

[0003] Currently, 5-HT and melatonin on the market are mainly synthesized by chemical methods. Chemical methods involve lengthy processes, cumbersome operations, limited overall yields, and high requirements for production equipment. While there are biotransformation synthesis methods for 5-HTP, its yield is still at a low level, only 14.9 g / L, and its industrialization process remains limited. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention is based on the biosynthetic pathway of melatonin (as shown in the appendix). Figure 1 As shown, the biosynthesis of different tryptophan derivatives such as 5-HTP, 5-HT and melatonin was achieved by introducing a cofactor module, strengthening the endogenous L-tryptophan synthesis pathway and adjusting the enzyme system components.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a phenylalanine hydroxylase mutant, XsP4H_m, obtained by mutating amino acid residues at three positions: W229F, L148I, and A179K, in the amino acid sequence shown in SEQ ID NO.1. The amino acid residue mutation pattern is represented in the form XnY, where X represents the original amino acid, n represents the mutation site, and Y represents the mutated amino acid; for example, W229F represents the mutation of amino acid W at position 229 of the amino acid sequence shown in SEQ ID NO.1 to F.

[0007] The aforementioned phenylalanine hydroxylase mutant XsP4H_m can be used in the synthesis of tryptophan derivatives, including 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), and N-acetyl-5-methoxytryptamine (melatonin).

[0008] The present invention further provides a recombinant engineered strain TRP-1, which is obtained by transforming an expression vector containing the above-mentioned phenylalanine hydroxylase mutant XsP4H_m, dehydratase (CvPcd), and reductase (EcFolM) encoding genes into a high-L-tryptophan-producing Escherichia coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL).

[0009] Specifically, the coding genes for XsP4H_m, CvPcd, and EcFolM were recombined into pET-30a to obtain the recombinant plasmid pET30a-xsp4h_m-cvpcd-ecfolm, which overexpressed these three genes in a polycistronic manner. The recombinant plasmid pET30a-xsp4h_m-cvpcd-ecfolm was then transformed into BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) to obtain the engineered strain TRP-1.

[0010] Based on the above, the present invention provides a method for synthesizing 5-hydroxytryptophan, using glucose as raw material and lactose or IPTG as an inducer, and using the above-mentioned recombinant engineered strain TRP-1 for fermentation to synthesize 5-hydroxytryptophan.

[0011] Specifically, the biotransformation synthesis of 5-HTP was carried out using the engineered strain TRP-1: glucose was used as the raw material, lactose or IPTG was used as the inducer, the pH was controlled at 6.9-7.1 during fermentation, the temperature was set at 37℃ before induction and 25℃ after induction, the dissolved oxygen level was controlled at 15-30%, and glucose was added to control the residual sugar concentration at 0.1-1g / L.

[0012] The present invention further provides a recombinant engineered strain TRP-2, which is obtained by transforming an expression vector containing the above-mentioned phenylalanine hydroxylase mutant XsP4H_m, dehydratase (CvPcd), reductase (EcFolM), and decarboxylase (CkDdc) encoding genes into a high-L-tryptophan-producing Escherichia coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL).

[0013] Specifically, the coding genes for XsP4H_m, CvPcd, and EcFolM were recombined into pET-30a to obtain the recombinant plasmid pET30a-xsp4h_m-cvpcd-ecfolm, which overexpressed these three genes in a polycistronic manner. The coding gene for CkDdc was recombined into pCDFDuet-1 to obtain the recombinant plasmid pCDF-ckddc. The recombinant plasmids pET30a-xsp4h_m-cvpcd-ecfolm and pCDF-ckddc were transformed into BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) to obtain the engineered strain TRP-2.

[0014] Based on the above, the present invention provides a method for synthesizing 5-hydroxytryptamine, using glucose as raw material and lactose or IPTG as an inducer, and using the above-mentioned recombinant engineered strain TRP-2 for fermentation to synthesize 5-hydroxytryptamine.

[0015] Specifically, the biotransformation synthesis of 5-HT was carried out using the engineered strain TRP-2: glucose was used as the raw material, lactose or IPTG was used as the inducer, the pH was controlled at 6.9-7.1 during fermentation, the temperature was set at 37℃ before induction and 25℃ after induction, the dissolved oxygen level was controlled at 15-30%, and glucose was added to control the residual sugar concentration at 0.1-1g / L.

[0016] The present invention further provides a recombinant engineered strain TRP-3, which is obtained by transforming an expression vector containing the above-mentioned phenylalanine hydroxylase mutant XsP4H_m, dehydratase (CvPcd), reductase (EcFolM), decarboxylase (CkDdc), acetyltransferase (SgAanat), and methyltransferase (HsAsmt) encoding genes into a high-L-tryptophan-producing Escherichia coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL).

[0017] Specifically, the coding genes for XsP4H_m, CkDdc, CvPcd, and EcFolM were recombined into pET-30a to obtain the recombinant plasmid pET30a-xsp4h_m-ckddc-cvpcd-ecfolm, which overexpressed these four genes in a polycistronic manner. The coding genes for SgAanat and HsAsmt were recombined into pCDFDuet-1 to obtain the recombinant plasmid pCDF-sgaanat-hsasmt. The recombinant plasmids pET30a-xsp4h_m-ckddc-cvpcd-ecfolm and pCDF-sgaanat-hsasmt were then transformed into BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) to obtain the engineered strain TRP-3.

[0018] Based on the above, the present invention provides a method for synthesizing N-acetyl-5-methoxytryptamine, using glucose as raw material and lactose or IPTG as an inducer, and using the above-mentioned recombinant engineered strain TRP-3 for fermentation to synthesize N-acetyl-5-methoxytryptamine.

[0019] Specifically, melatonin was synthesized by biotransformation using engineered strain TRP-3: glucose was used as raw material, lactose or IPTG was used as an inducer, the pH was controlled at 6.9-7.1 during fermentation, the temperature was set at 37℃ before induction and at 25℃ after induction, the dissolved oxygen level was controlled at 15-30%, and glucose was added to control the residual sugar concentration at 0.1-1g / L.

[0020] The aforementioned recombinant engineered strains TRP-1, TRP-2, and TRP-3 can be used in the synthesis of tryptophan derivatives such as 5-HTP, 5-HT, and melatonin.

[0021] The amino acid sequences of CvPcd, EcFolM, CkDdc, SgAanat, and HsAsmt are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively; the DNA sequences encoding CvPcd, EcFolM, CkDdc, SgAanat, and HsAsmt are shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0022] The construction method of the above-mentioned high-L-tryptophan-producing Escherichia coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) is as follows:

[0023] Using genome editing, the genes tnaA, trpR, TyrA, pheA, and TrpL on the genome of Escherichia coli BL21(DE3) were knocked out sequentially to construct a high-L-tryptophan-producing Escherichia coli strain BL21(ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL).

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention utilizes genome editing to construct a high-L-tryptophan-producing Escherichia coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL). Using this strain as a chassis cell, the following enzymes are overexpressed in different combinations: a mutant of phenylalanine hydroxylase (XsP4H_m), dehydratase (CvPcd), reductase (EcFolM), decarboxylase (CkDdc), acetyltransferase (SgAanat), and methyltransferase (HsAsmt). Tryptophan derivatives such as 5-HTP, 5-HT, or melatonin are synthesized using bio-fermentation methods. The efficient biosynthesis of tryptophan derivatives can be achieved at room temperature and normal pressure.

[0026] The engineered bacteria constructed in this invention exhibited high production capacity during batch-fed fermentation in a 5L fermenter. Engineered strain TRP-1, fermented at 25°C for 40 hours, synthesized 26.9 g / L of 5-HTP, representing the highest yield of 5-HTP synthesized using biological methods to date. Engineered strain TRP-2, fermented at 25°C for 40 hours, synthesized 8.9 g / L of 5-HT without producing the byproduct tryptamine (TRPM). Engineered strain TRP-3, fermented at 30°C for 60 hours, synthesized 2.1 g / L of melatonin. Attached Figure Description

[0027] Figure 1 This describes the biosynthetic route of melatonin.

[0028] Figure 2 HPLC analysis of the fermentation broth of engineered strain TRP-1.

[0029] Figure 3 HPLC analysis of the fermentation broth of engineered strain TRP-2.

[0030] Figure 4 HPLC analysis of the fermentation broth of engineered strain TRP-3. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] This invention provides a phenylalanine hydroxylase mutant XsP4H_m, which is obtained by performing a three-point mutation W229F / L148I / A179K on XsP4H. The amino acid sequence of XsP4H is shown in SEQ ID NO.1; the DNA sequence encoding XsP4H is shown in SEQ ID NO.7.

[0033] Furthermore, this invention utilizes genome editing techniques to construct a high-L-tryptophan-producing Escherichia coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL), and uses this strain as a chassis cell to overexpress the following enzymes in different combinations: a mutant of phenylalanine hydroxylase (XsP4H_m), dehydratase (CvPcd), reductase (EcFolM), decarboxylase (CkDdc), acetyltransferase (SgAanat), and methyltransferase (HsAsmt). Then, it uses bio-fermentation methods to synthesize tryptophan derivatives such as 5-HTP, 5-HT, or melatonin.

[0034] Example 1: Construction of recombinant plasmid pET30a-xsp4h_m-cvpcd-ecfolm

[0035] Using the synthesized XsP4H gene (amino acid sequence as described in SEQ ID NO: 1) as a template, the corresponding target genes xsp4h, cvpcd, and ecfolm were amplified by PCR using KOD one DNA polymerase and primer pairs F1 / R1, F2 / R2, and F3 / R3 (Table 1). The amplification program was: 98℃, 3 min; 98℃, 10 s, 60℃, 20 s, 68℃, 10 s, 30 cycles; 68℃, 2 min. The target fragments were recovered using a gel extraction kit. Using the recovered xsp4h, cvpcd, and ecfolm fragments as templates, overlap extension PCR was performed using primer F1 / R3 to amplify the fusion fragment xsp4h-cvpcd-ecfolm. The target fragment was recovered using a gel extraction kit. The pET30a plasmid and the xsp4h-cvpcd-ecfolm fusion fragment were double-digested with BamHI and XhoⅠ restriction endonucleases, respectively, and the target fragment was recovered using a gel extraction kit. The xsp4h-cvpcd-ecfolm fragment was ligated into the linearized vector pET30a using T4 ligase to construct the pET30a-xsp4h-cvpcd-ecfolm recombinant plasmid. The enzyme ligation product was then heat-shocked and transformed into *E. coli* DH5α competent cells, and plated on LB agar plates containing 50 mg / L kanamycin. Single colonies from the selection plate were picked, cultured in 25 mL of LB liquid medium containing kanamycin, and then the plasmid was extracted. Sequencing analysis confirmed the sequence was correct, and the plasmid was considered the recombinant plasmid pET30a-xscp4h-cvpcd-ecfolm.

[0036] The expression plasmid of the mutant was amplified by circular plasmid PCR using the recombinant plasmid pET30a-xsp4h-cvpcd-ecfolm and KOD one DNA polymerase. The primers used are shown in Table 1. The amplification program was as follows: 98℃, 3 min; 98℃, 10 s, 60℃, 20 s, 68℃, 40 s, 30 cycles; 68℃, 2 min. After PCR, 0.3 U DMT was added to the reaction system, and the reaction was carried out at 37℃ for 1 h to digest the template. After digestion, 3 μL of the digestion product was transferred into E. coli BL21(DE3) competent cells and then plated on LB agar plates containing 50 mg / L kanamycin.

[0037] After successful sequencing confirmation of the mutation, the plasmid was extracted as a template for a new round of site-directed mutagenesis, and finally the recombinant plasmid pET30a-xsp4h_m-cvpcd-ecfolm was obtained, carrying the XsP4H three-point mutant XsP4H-W229F / L148I / A179K (XsP4H_m).

[0038] Using the same method, recombinant plasmids pET30a-xsp4h_m-ckddc-cvpcd-ecfolm, pCDF-ckddc, and pCDF-sgaanat-hsasmt were constructed.

[0039] Table 1. Primer Information

[0040]

[0041] Example 2: Genome Editing of Chassis Cells

[0042] Taking tnaA knockout as an example, this paper introduces the process of editing the chassis cell genome.

[0043] 1) Construction of the target plasmid pTarget F-tnaA-gRNA

[0044] Using plasmid pTarget F as a template, the targeting plasmid pTarget F-tnaA-gRNA was constructed by circular PCR using primer pair tnaA-F / tnaA-R (Table 1). The obtained PCR product was digested with DMT (37℃, 1h) and then heat-shocked into E. coli DH5α competent cells. Transformants were picked, cultured, and plasmids were extracted. DNA sequencing confirmed the successful construction of the targeting plasmid pTarget F-tnaA-gRNA.

[0045] 2) Amplification of repair template

[0046] Using E. coil BL21(DE3) bacterial culture as a template, the upstream and downstream homologous arms of the tnaA gene were amplified using primer pairs tnaA-UF / tnaA-UR and tnaA-DF / tnaA-DR (Table 1). After purification and recovery, the fusion fragment of the upstream and downstream homologous arms was amplified by PCR using the two fragments as templates and primer pairs tnaA-UF / tnaA-DR. After purification and recovery, a repair template of 1091 bp was obtained.

[0047] 3) Conversion and Screening

[0048] The targeting plasmid pTarget F-tnaA-gRNA and the repair template were simultaneously transformed into E. coil BL21(DE3) competent cells carrying the pEcCas plasmid using electroporation. The cells were then plated on LB agar plates containing 50 mg / L kanamycin, 50 mg / L streptomycin and 10 mM arabinose and cultured at 37°C for 12-16 h until single colonies appeared.

[0049] Single colonies were picked from the plate, and E. coil BL21(DE3) genome was used as a control. Colony PCR was performed using DNA polymerase TaqPCR Master with primer pair tnaA-UF / tnaA-DR. The PCR products were subjected to agarose gel electrophoresis. The control group band size was 2507 bp. If the transformant band size was a fragment of about 1091 bp, it indicated that the tnaA gene knockout of the transformant was successful.

[0050] 4) Elimination of gene-editing elements

[0051] Transformants with successful tnaA gene knockout were selected and inoculated into 1 mL of LB liquid medium containing kanamycin and 10 mM rhamnose. They were incubated at 37°C and 200 rpm for 12–16 h to eliminate the target plasmid pTarget F-tnaA-gRNA. The strain was identified as BL21(ΔtnaA)-pEcCas. These were then converted into competent cells for use in the next gene editing process.

[0052] Using the same method, the genes pheA, tyrA, trpR, and trpL were sequentially knocked out to obtain the engineered strain BL21(ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL)-pEcCas. This strain was inoculated into 1 mL of LB liquid medium containing 5 g / L glucose and cultured at 37°C and 200 rpm for 12–16 h to eliminate the pEcCas plasmid. The culture was then diluted 10⁻⁶ times. 6 After dilution, the samples were plated onto LB agar containing 5 g / L glucose and 10 g / L sucrose and incubated at 37°C for 12–16 h. The kanamycin-sensitive clones were the plasmid-depleted gene-edited strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL).

[0053] Example 3: Construction of an engineered strain producing high levels of 5-HTP

[0054] The recombinant plasmid pET30a-xsp4h_m-cvpcd-ecfolm was transformed into competent cells of the gene-edited strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) using a heat shock transformation method. The cells were then plated on LB agar plates containing 50 mg / L kanamycin and cultured at 37°C for 12-16 h until a single colony grew. This resulted in the engineered strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL, pET30a-xsp4h_m-cvpcd-ecfolm) that produces high levels of 5-hydroxytryptophan, and was named TRP-1.

[0055] Tests showed that the shake-flask fermentation yield of strain TRP-1 was 68% higher than that of the wild-type high-5-hydroxytryptophan-producing engineered strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL, pET30a-xsp4h-cvpcd-ecfolm) (0.52 g / L vs. 0.31 g / L).

[0056] Example 4: Fermentation Synthesis of 5-HTP

[0057] The engineered strain TRP-1 was activated on LB agar plates containing 50 mg / L kanamycin. After single colonies grew, a single colony of the engineered strain was picked and inoculated into 150 mL of LB medium containing kanamycin. The culture was carried out at 37 °C and 200 rpm for 12 h to obtain the seed culture. The entire seed culture was then inoculated into a 5 L fermenter (3 L of liquid, 2 g / L anhydrous glucose, 10 g / L glycerol, 15 g / L yeast extract, 2.5 g / L (NH4)2SO4, 4 g / L K2HPO4, 2.25 g / L NaH2PO4, 3 g / L NaCl, 2.1 g / L citric acid monohydrate, 0.67 g / L MgSO4·7H2O, and 66 mg / L FeSO4·7H2O) to start fermentation. The stirring speed was set to 300 rpm, and the temperature to 37℃. During fermentation, the pH was controlled at around 7.0 by automatically adding NH4OH (25%, v / v). When nutrients were depleted, dissolved oxygen and pH spiked. At this point, a feed start-up (600 g / L glucose) was initiated to maintain residual sugar within the range of 0.1-1 g / L. Dissolved oxygen was maintained at around 30% by adjusting the stirring speed. When OD... 600 When the concentration reached approximately 20, IPTG was added to a final concentration of 0.2 mM to induce the expression of functional genes. Samples were taken during fermentation and the 5-HTP content was analyzed using HPLC. The HPLC mobile phase was: water (pH 3.4) / acetonitrile = 94 / 6; temperature: 30℃; sample loading volume: 20 μL; flow rate: 1 mL / min; detection wavelength: 276 nm; column: Elite Supersil ODS2 5 μm C18 (4.6 mm × 250 mm). After 40 h of fermentation, the yield reached 26.9 g / L, with a space-time yield of 0.67 g / L / h, which is the highest reported level for 5-HTP production by engineered E. coli fermentation to date.

[0058] Example 5: Construction of engineered bacteria producing high levels of 5-HT

[0059] The recombinant plasmids pET30a-xsp4h_m-cvpcd-ecfolm and pCDF-ckddc were transformed into competent cells of the gene-edited strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) using a heat shock co-transformation method. The cells were then plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L streptomycin and cultured at 37°C for 12-16 h until a single colony grew, which was the high-5-HT-producing engineered strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL, pET30a-xsp4h_m-cvpcd-ecfolm, pCDF-ckddc), and named TRP-2.

[0060] Example 6: Fermentation Synthesis of 5-HT

[0061] The engineered strain TRP-2 was activated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L streptomycin. After single colonies grew, a single colony was picked and inoculated into 150 mL of LB medium containing kanamycin and streptomycin. The culture was incubated at 37°C and 200 rpm for 12 h to obtain the seed culture. This seed culture was then inoculated into a 5 L fermenter (medium as in Example 4) to start fermentation. The stirring speed was set to 300 rpm, and the temperature to 37°C. During fermentation, the pH was controlled at approximately 7.0 by automatically adding NH4OH (25%, v / v). When nutrients were depleted, dissolved oxygen and pH spiked. At this point, feeding (600 g / L glucose) was initiated to maintain residual sugar in the range of 0.1-1 g / L. Dissolved oxygen was maintained at approximately 30% by adjusting the stirring rate. When OD... 600 When the concentration reached approximately 20, IPTG was added to a final concentration of 0.2 mM to induce the expression of functional genes. Samples were taken during fermentation and the 5-HT content was analyzed using HPLC. The HPLC mobile phase was: water (pH 3.4) / acetonitrile = 96 / 4; temperature: 30℃; sample volume: 20 μL; flow rate: 1 mL / min; detection wavelength: 276 nm; column: ShimNex CS C18 5 μm (4.6 mm × 250 mm). After 40 h of accumulation, 8.9 g / L 5-HT was obtained.

[0062] Example 7: Construction of melatonin-producing engineered bacteria

[0063] The recombinant plasmids pET30a-xsp4h_m-ckddc-cvpcd-ecfolm and pCDF-sgaanat-hsasmt were transformed into competent cells of the gene-edited strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) via heat shock co-transformation. The cells were then plated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L streptomycin and cultured at 37°C for 12-16 h until a single colony appeared. This resulted in the high-melatonin-producing engineered strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL, pET30a-xsp4h_m-ckddc-cvpcd-ecfolm, pCDF-sgaanat-hsasmt), named TRP-3.

[0064] Example 8: Fermentation Synthesis of Melatonin

[0065] The engineered strain TRP-3 was activated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L streptomycin. After single colonies grew, a single colony of the engineered strain was picked and inoculated into 150 mL of LB medium containing kanamycin and streptomycin. The culture was carried out at 37°C and 200 rpm for 12 h to obtain the seed culture. This seed culture was then inoculated into a 5 L fermenter (medium as in Example 4) to start fermentation. The stirring speed was set to 300 rpm, and the temperature to 37°C. During fermentation, the pH was controlled at approximately 7.0 by automatically adding NH4OH (25%, v / v). When nutrients were depleted, dissolved oxygen and pH spiked. At this point, feeding (600 g / L glucose) was initiated to maintain residual sugar in the range of 0.1-1 g / L. Dissolved oxygen was maintained at approximately 30% by adjusting the stirring rate. When OD... 600 When the concentration reached approximately 20, IPTG was added to a final concentration of 0.2 mM to induce the expression of functional genes. Melatonin content was detected by HPLC using samples taken during fermentation. Mobile phase: water (pH 3.4) / acetonitrile = 70 / 30; temperature: 30℃; injection volume: 20 μL; flow rate: 1 mL / min; detection wavelength: 278 nm; column: ShimNex CS C18 5 μm (4.6 mm × 250 mm). After 60 h of accumulation, 2.1 g / L of melatonin was obtained.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A phenylalanine hydroxylase mutant, characterized in that, The phenylalanine hydroxylase mutant was obtained by mutating amino acid residues at three positions: W229F, L148I, and A179K, in the amino acid sequence shown in SEQ ID NO.

1.

2. The use of the phenylalanine hydroxylase mutant as described in claim 1 in the synthesis of 5-hydroxytryptophan, 5-hydroxytryptamine or N-acetyl-5-methoxytryptamine.

3. A recombinant engineered strain TRP-1, characterized in that, The recombinant engineered strain TRP-1 was obtained by transforming a high-L-tryptophan-producing Escherichia coli strain into an expression vector containing the phenylalanine hydroxylase mutant, CvPcd, and EcFolM encoding genes as described in claim 1. The encoding genes for CvPcd and EcFolM are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively; The method for constructing the Escherichia coli strain with high L-tryptophan production is as follows: Using genome editing, the following genes were sequentially knocked out in the genome of E. coli BL21(DE3). tnaA , trpR , TyrA , pheA and TrpL Genes were used to construct a high-L-tryptophan-producing Escherichia coli strain BL21(Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL ).

4. A method for synthesizing 5-hydroxytryptophan, characterized in that, Using glucose as a raw material and lactose or IPTG as an inducer, 5-hydroxytryptophan was synthesized by fermentation using the recombinant engineered strain TRP-1 as described in claim 3.

5. The application of the recombinant engineered strain TRP-1 as described in claim 3 in the synthesis of 5-hydroxytryptophan.

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