Recombinant engineering strain TRP-3 and application thereof in melatonin synthesis
By constructing the recombinant engineered strain TRP-3 and utilizing gene editing and enzyme system component adjustment, the efficient biosynthesis of melatonin was achieved, solving the problems of lengthy chemical synthesis processes and high equipment requirements, and improving production efficiency and total yield.
Patent Information
- Application Number
- CN202511318230.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-29
- Publication Date
- 2025-12-16
AI Technical Summary
Existing chemical synthesis processes for melatonin are lengthy, cumbersome, have limited overall yields, and require sophisticated production equipment.
By constructing the recombinant engineered strain TRP-3, a high-L-tryptophan-producing Escherichia coli strain BL21 was constructed using genome editing methods. The phenylalanine hydroxylase mutant XsP4H_m, dehydratase CvPcd, reductase EcFolM, decarboxylase CkDdc, acetyltransferase SgAanat, and methyltransferase HsAsmt were overexpressed to achieve the biosynthesis of melatonin.
The efficient biosynthesis of melatonin was achieved at room temperature and normal pressure, improving production efficiency and overall yield.
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Abstract
Description
[0001] Divisional application statement This application is a divisional application of Chinese Patent Application No. 202411020959.1, filed on July 29, 2024, entitled "A phenylalanine hydroxylase mutant and its application in the synthesis of tryptophan derivatives". Technical Field
[0002] This invention belongs to the fields of biocatalysis and biopharmaceuticals, specifically relating to a recombinant engineered strain TRP-3 and its application in melatonin synthesis. Background Technology
[0003] Melatonin (N-acetyl-5-methoxytryptamine) is a biogenic amine that has become increasingly well-known for its anti-inflammatory, antioxidant, and autophagy-inducing 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.
[0004] Currently, melatonin on the market is mainly synthesized by chemical methods. The chemical process is lengthy, complicated, has a limited overall yield, and requires high-quality production equipment. Summary of the Invention
[0005] 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, melatonin is synthesized biologically through the introduction of a cofactor module, the enhancement of the endogenous L-tryptophan synthesis pathway, and the adjustment of enzyme components.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: This invention provides a recombinant engineered bacterial strain TRP-3, which is transformed into a high-L-tryptophan-producing *Escherichia coli* strain BL21(Δ) using an expression vector containing genes encoding a phenylalanine hydroxylase mutant XsP4H_m, a dehydratase (CvPcd), a reductase (EcFolM), a decarboxylase (CkDdc), an acetyltransferase (SgAanat), and a methyltransferase (HsAsmt). tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL Obtained in ) 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.
[0007] Specifically, the coding genes of XsP4H_m, CkDdc, CvPcd and EcFolM are recombined into pET-30a to obtain a recombinant plasmid pET30a- xsp4h_m - ckddc - cvpcd - ecfolm SgAanat and HsAsmt are recombined into pCDFDuet-1 to obtain a recombinant plasmid pCDF- sgaanat - hsasmt The recombinant plasmids pET30a- xsp4h_m - ckddc-cvpcd-ecfolm and pCDF- sgaanat-hsasmt are transformed into BL21 (Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL ) to obtain an engineering strain TRP-3.
[0008] Further, the coding genes of CvPcd, EcFolM, CkDdc, SgAanat and HsAsmt are respectively shown as SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.
[0009] Further, the construction method of the high-yield L-tryptophan E. coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) is as follows: By using the method of genome editing, the genes of tnaA 、 trpR 、 TyrA 、 pheA and TrpL on the genome of E. coli BL21 (DE3) are sequentially knocked out to construct the high-yield L-tryptophan E. coli strain BL21 (Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL .
[0010] Based on the above, the present application provides a synthesis method of melatonin, taking glucose as raw material, lactose or IPTG as inducer, and using the above-mentioned recombinant engineering strain TRP-3 to ferment and synthesize melatonin, i.e. N-acetyl-5-methoxytryptamine.
[0011] Specifically, the biosynthesis of melatonin is carried out by using the engineering strain TRP-3: glucose is used as raw material, lactose or IPTG is used as inducer, the pH in the fermentation process is controlled to be 6.9-7.1, the temperature before induction is set to be 37 DEG C, the temperature after induction is set to be 25 DEG C, the dissolved oxygen level is controlled to be 15-30%, and the glucose is added in flow to control the residual sugar concentration to be 0.1-1 g / L.
[0012] The above-mentioned recombinant engineering strain TRP-3 can be applied to the biosynthesis of melatonin.
[0013] Compared with the prior art, the present application has the following beneficial effects: The present application uses a genome editing method to construct an E. coli strain BL21 (Delta tnaA / Delta trpR / Delta TyrA / Delta pheA / Delta TrpL ) with high yield of L-tryptophan, and uses the strain as a chassis cell to overexpress the following enzymes: mutant of phenylalanine hydroxylase (XsP4H_m), dehydrase (CvPcd), reductase (EcFolM), decarboxylase (CkDdc), acetyltransferase (SgAanat) and methyltransferase (HsAsmt), and uses a biological fermentation method to synthesize the derivative of tryptophan, melatonin, so that the high-efficiency biosynthesis of the derivative of tryptophan, melatonin (N-acetyl-5-methoxytryptamine), can be realized at room temperature and under normal pressure. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 The above-mentioned recombinant engineering strain TRP-3 can be applied to the biosynthesis of melatonin.
[0015] Figure 2 The above-mentioned recombinant engineering strain TRP-3 can be applied to the biosynthesis of melatonin.
[0016] Figure 3 The above-mentioned recombinant engineering strain TRP-3 can be applied to the biosynthesis of melatonin.
[0017] Figure 4 The above-mentioned recombinant engineering strain TRP-3 can be applied to the biosynthesis of melatonin. DETAILED DESCRIPTION
[0018] To make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.
[0019] 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.
[0020] Furthermore, the present invention utilizes genome editing techniques to construct a high-L-tryptophan-producing Escherichia coli strain BL21(Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL Using this strain as the chassis cell, the following enzymes were overexpressed in different combinations: mutant 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 were synthesized by bio-fermentation.
[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] Example 1: Recombinant plasmid pET30a- xsp4h_m - cvpcd - ecfolm Construction Using the synthesized XsP4H (amino acid sequence as described in SEQ ID NO: 1) encoding gene (sequence as described in SEQ ID NO: 7) as a template, the corresponding target gene was amplified by PCR using KOD one DNA polymerase and primer pairs F1 / R1, F2 / R2, and F3 / R3 (Table 1). xsp4h , cvpcd , ecfolm The amplification program was as follows: 98℃, 3 min; 98℃, 10 s, 60℃, 20 s, 68℃, 10 s, 30 cycles; 68℃, 2 min. The target fragment was recovered using a gel recovery kit. The recovered fragment...xsp4h 、 cvpcd 、 ecfolm The fusion fragment was amplified by overlap extension PCR using the primers F1 / R3 xsp4h - cvpcd - ecfolm The target fragment was recovered using a gel recovery kit. The pET30a plasmid and the fusion fragment were double-digested with BamH I and Xho I restriction enzymes, respectively. xsp4h - cvpcd - ecfolm The target fragment was recovered using a gel recovery kit. The pET30a plasmid and the fusion fragment were double-digested with BamH I and Xho I restriction enzymes, respectively. xsp4h - cvpcd - ecfolm The fusion fragment was amplified by overlap extension PCR using the primers F1 / R3 xsp4h - cvpcd - ecfolm The recombinant plasmid was then heat-shocked and transformed into E. coli DH5α competent cells, which were then plated on LB plates containing 50 mg / L of kanamycin resistance. Single colonies were selected from the screening plate, and the plasmid was extracted after the colonies were cultured in 25 mL of LB liquid medium containing kanamycin. The sequence was correct after sequencing analysis, and the recombinant plasmid pET30a- xscp4h - cvpcd - ecfolm .
[0023] The recombinant plasmid pET30a- xsp4h - cvpcd - ecfolm The expression plasmid of the mutant was amplified by circular plasmid PCR using KOD one DNA polymerase. The primers used are shown in Table 1. The amplification program was as follows: 98°C, 3 min; 98°C, 10 s, 60°C, 20 s, 68°C, 40 s, for 30 cycles; 68°C, 2 min. After the PCR was completed, 0.3 U of DMT was added to the reaction system, and the template was digested at 37°C for 1 h. After the digestion was completed, 3 µL of the digestion product was transferred into E.coli BL21 (DE3) competent cells, which were then plated on LB plates containing 50 mg / L of kanamycin resistance.
[0024] After the successful mutation was confirmed by sequencing, 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 .
[0025] Using the same method, recombinant plasmid pET30a- xsp4h_m - ckddc - cvpcd - ecfolm , pCDF- ckddc and pCDF- sgaanat - hsasmt .
[0026] Table 1. Primer information
[0027] Example 2: Genome editing of chassis cells Take the knockout of tnaA as an example to introduce the process of editing the genome of chassis cells.
[0028] 1) Construction of targeting plasmid pTarget F- tnaA -gRNA Using the plasmid pTarget F as a template, use primer pair tnaA -F / tnaA -R (Table 1) to perform whole plasmid circular PCR to construct the targeting plasmid pTarget F- tnaA -gRNA. The resulting PCR product is digested with DMT (37°C, 1 h) and then heat-shocked into E. coli DH5α competent cells. The transformants are picked for culture and plasmid extraction, and DNA sequencing confirms that the targeting plasmid pTarget F- tnaA -gRNA is successfully constructed.
[0029] 2) Amplification of repair template Using the E. coil BL21 (DE3) bacterial solution as a template, use primer pair tnaA -U-F / tnaA -U-R and tnaA -D-F / tnaA -D-R (Table 1) to amplify the upstream and downstream homologous arms of the tnaA gene. After purification and recovery, use the two fragments as templates and primer pair tnaA -U-F / tnaA -D-R to PCR amplify the fusion fragment of the upstream and downstream homologous arms. After purification and recovery, a repair template with a size of 1091 bp is obtained.
[0030] 3) Transformation and selection Using the method of electroporation, the targeting plasmid pTarget F- tnaA -gRNA and the repair template are simultaneously transformed into E. coilBL21(DE3) competent cells were then spread onto LB agar plates containing 50 mg / L kanamycin, 50 mg / L streptomycin and 10 mM arabinose and incubated at 37°C for 12-16 h until single colonies appeared.
[0031] Pick a single colony from the plate, to E.coil Using the BL21(DE3) genome as a control, DNA polymerase Taq PCRMaster was used with primer pairs. tnaA -UF / tnaA -DR was used for colony PCR, and 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 transformant was a healthy organism. tnaA Gene knockout was successful.
[0032] 4) Elimination of gene-editing elements Pick tnaA Transformants that successfully knocked out the gene were inoculated into 1 mL of LB liquid medium containing kanamycin and 10 mM rhamnose and cultured at 37°C and 200 rpm for 12–16 h to eliminate the target plasmid pTarget F- tnaA -gRNA. This strain is BL21 (Δ tnaA )-pEcCas. Reconstitute it into a competent state for use in the next gene editing task.
[0033] Continue knocking out using the same method. pheA , tyrA , trpR and trpL By using the same genes, engineered bacteria BL21(Δ) were obtained. tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL )-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 The plasmid-free gene-edited strain BL21 (Δ) was then 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 clone was the plasmid-depleted gene-edited strain. tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL ).
[0034] Example 3: Construction of an engineered strain producing high levels of 5-HTP The recombinant plasmid pET30a- xsp4h_m - cvpcd - ecfolm The competent cells of the gene editing strain BL21 (Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL ) were transformed by heat shock transformation method, then coated on LB plates containing 50 mg / L kanamycin, cultured at 37°C for 12-16 h until single colonies grew, which was the high-yield 5-hydroxytryptophan engineering strain BL21 (Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL , pET30a- xsp4h_m - cvpcd - ecfolm ), named TRP-1.
[0035] Tested, the yield of TRP-1 strain in shake flask fermentation was increased by 68% (0.52 g / L vs. 0.31 g / L) compared with the wild type high-yield 5-hydroxytryptophan engineering strain BL21 (Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL , pET30a- xsp4h - cvpcd - ecfolm ).
[0036] Example 4: Fermentative synthesis of 5-HTP The engineering strain TRP-1 was activated on LB plate containing 50 mg / L kanamycin, and after a single colony grew, the single colony of the engineering bacteria was picked and inoculated into 150 mL LB medium containing kanamycin, and cultured at 37°C, 200 rpm for 12 h as a seed liquid. The seed liquid was inoculated into a 5 L fermenter (liquid volume 3 L, anhydrous glucose 2 g / L, glycerol 10 g / L, yeast extract powder 15 g / L, (NH4)2SO4 2.5 g / L, K2HPO4 4 g / L, NaH2PO4 2.25 g / L, NaCl 3 g / L, citric acid monohydrate 2.1 g / L, MgSO4·7H2O 0.67 g / L, FeSO4·7H2O 66 mg / L), and the fermentation was started. The stirring speed was set to 300 rpm, and the temperature was set to 37°C. During the fermentation process, the pH value was controlled at about 7.0 by automatically adding NH4OH (25%, v / v). When the nutrients were exhausted, the dissolved oxygen and pH rose, at which time the feeding (600 g / L glucose) was started to maintain the residual sugar in the range of 0.1-1 g / L. The dissolved oxygen was maintained at about 30% by adjusting the stirring rate. When the OD 600 The fermentation process was sampled and analyzed for 5-HTP content by HPLC. The mobile phase of HPLC: water (pH 3.4) / acetonitrile=94 / 6; temperature: 30°C; sample volume: 20 uL; flow rate: 1 mL / min; detection wavelength: 276 nm; chromatographic column: Elite Supersil ODS2 5 μm C18 (4.6 mm x 250 mm). After 40 h of fermentation, the yield reached 26.9 g / L, and the space-time yield was 0.67 g / L / h, which was the highest level reported in the fermentation production of 5-HTP by engineering Escherichia coli.
[0037] Example 5: Construction of engineering bacteria with high 5-HT production The recombinant plasmids pET30a- xsp4h_m - cvpcd - ecfolm and pCDF- ckddc were transformed into the competent cells of the gene editing strain BL21 (Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL ) by heat shock co-transformation method, and then coated on LB plate 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 engineering strain BL21 (Δ tnaA / Δ trpR / ΔTyrA / Δ pheA / Δ TrpL , pET30a- xsp4h_m - cvpcd - ecfolm , pCDF- ckddc ), named as TRP-2.
[0038] Example 6: Fermentative synthesis of 5-HT The engineered strain TRP-2 was activated on LB plate containing 50 mg / L kanamycin and 50 mg / L streptomycin. After single colony appeared, the single colony of the engineered strain was picked and inoculated into 150 mL LB medium containing kanamycin and streptomycin. The culture was incubated at 37 °C, 200 rpm for 12 h as seed liquid. The seed liquid was inoculated into 5 L fermenter (medium as in Example 4) and fermentation was started. The stirring speed was set at 300 rpm and the temperature was set at 37 °C. During the fermentation, the pH was controlled at about 7.0 by automatic addition of NH4OH (25%, v / v). When the nutrients were exhausted, the dissolved oxygen and pH rose sharply, at which time the feeding (600 g / L glucose) was started to maintain the residual sugar in the range of 0.1-1 g / L. The dissolved oxygen was maintained at about 30% by adjusting the stirring speed. When the OD600 reached about 20, IPTG was added to a final concentration of 0.2 mM to induce the expression of functional genes. The content of 5-HT was analyzed by HPLC during the fermentation. The mobile phase of HPLC: water (pH 3.4) / acetonitrile = 96 / 4; temperature: 30 °C; sample volume: 20 uL; flow rate: 1 mL / min; detection wavelength: 276 nm; column: ShimNex CS C18 5 μm (4.6 mm x 250 mm). After 40 h of accumulation, 8.9 g / L 5-HT was obtained. 600
[0039] Example 7: Construction of engineered strain for melatonin production Recombinant plasmid pET30a- xsp4h_m - ckddc - cvpcd - ecfolm and pCDF- sgaanat - hsasmt The gene editing strain BL21 (Δ tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL ) was transformed into the competent cells by heat shock co-transformation method, and then plated on LB plate containing 50 mg / L kanamycin and 50 mg / L streptomycin and incubated at 37 °C for 12-16 h until single colonies appeared, which was the engineered strain BL21 (ΔtnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL , pET30a- xsp4h_m - ckddc - cvpcd - ecfolm , pCDF- sgaanat - hsasmt ), named TRP-3.
[0040] Example 8: Fermentative synthesis of melatonin The engineering strain TRP-3 was activated on LB plate containing 50 mg / L kanamycin and 50 mg / L streptomycin, and after the single colony grew, the single colony of engineering bacteria was picked and inoculated into 150 mL LB medium containing kanamycin and streptomycin, and cultured at 37℃, 200 rpm for 12 h as seed liquid, and the seed liquid was inoculated into 5 L fermenter (the medium was the same as that in Example 4), and the fermentation was started. The stirring speed was set to 300 rpm, and the temperature was set to 37℃, and during the fermentation, NH4OH (25%, v / v) was automatically added to control the pH value at about 7.0. When the nutrients were exhausted, the dissolved oxygen and pH value rose, at which time the feeding (600 g / L glucose) was started, and the residual sugar was maintained in the range of 0.1-1 g / L. By adjusting the stirring speed, the dissolved oxygen was maintained at about 30%. When the OD 600 reached about 20, IPTG with a final concentration of 0.2 mM was added to induce the expression of functional genes. The melatonin content was detected by HPLC during the fermentation process. The mobile phase: water (pH 3.4) / acetonitrile=70 / 30, temperature: 30℃; injection volume: 20 uL; flow rate: 1 mL / min; detection wavelength: 278 nm; chromatographic column: ShimNex CS C18 5 μm (4.6 mm×250 mm). After 60 h of accumulation, 2.1 g / L melatonin was obtained.
[0041] In summary, the engineering strain constructed in the application has high production capacity in the fed-batch fermentation process in a 5 L fermenter. The engineering strain TRP-1 can synthesize 26.9 g / L 5-HTP under the condition of fermentation at 25℃ for 40 h, which is the highest yield of 5-HTP synthesized by biological method at present. The engineering strain TRP-2 can synthesize 8.9 g / L 5-HT under the condition of fermentation at 25℃ for 40 h, and no by-product tryptamine (TRPM) is produced. The engineering strain TRP-3 can synthesize 2.1 g / L melatonin under the condition of fermentation at 30℃ for 60 h.
[0042] The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements 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 application.
Claims
1. A recombinant engineered bacterial strain TRP-3, characterized in that, The recombinant engineered strain TRP-3 was obtained by transforming a high-L-tryptophan-producing Escherichia coli strain into an expression vector containing the gene encoding phenylalanine hydroxylase mutant, CvPcd, EcFolM, CkDdc, SgAanat, and HsAsmt. 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 recombinant engineered strain TRP-3 according to claim 1, characterized in that, The encoding genes for 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.
3. The recombinant engineered strain TRP-3 according to claim 1, characterized in that, 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 melatonin, characterized in that, Melatonin is synthesized by fermentation using glucose as raw material and lactose or IPTG as an inducer, based on the recombinant engineered strain TRP-3 as described in any one of claims 1 to 3.
5. The method for synthesizing melatonin according to claim 4, characterized in that, Using glucose as raw material and lactose or IPTG as an 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 0.1-1 g / L.
6. The application of the recombinant engineered strain TRP-3 as described in any one of claims 1 to 3 in melatonin synthesis.