Recombinant engineering strain TRP-2 and application thereof in serotonin synthesis

Abstract

The invention belongs to the field of biological catalysis and biological pharmacy, and particularly relates to a recombinant engineering strain TRP-2 and application thereof in serotonin synthesis. According to the invention, an escherichia coli strain BL21 ([delta] tnaA / [delta] trpR / [delta] TyrA / [delta] pheA / [delta] TrpL) with high yield of L-tryptophan is constructed by using a genome editing means, the strain is taken as a chassis cell, the following enzymes are over-expressed: a mutant (XsP4Hm) of phenylalanine hydroxylase, dehydratase (CvPcd), reductase (EcFolM) and decarboxylase (CkDdc), and serotonin (5-hydroxytryptamine) is synthesized by using a biological fermentation method. And efficient biosynthesis of the serotonin is realized at room temperature and normal pressure.

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-2 and its application in serotonin synthesis. Background Technology

[0003] 5-Hydroxytryptamine (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 a variety of diseases, including depression, anxiety, hypertension, and irritable bowel syndrome.

[0004] Currently, serotonin on the market is mainly synthesized through chemical methods. These methods involve lengthy and complex processes, limited overall yields, and demanding production equipment. In recent years, the implementation of environmental policies has forced companies to seek greener methods for serotonin synthesis. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention is based on the biosynthetic pathway of serotonin (as shown in the appendix). Figure 1 As shown, serotonin is synthesized biosynthetically 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-2, 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), and a decarboxylase (CkDdc). 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 for XsP4H_m, CvPcd, and EcFolM were recombined into pET-30a to obtain the recombinant plasmid pET30a- xsp4h_m - cvpcd - ecfolm These three genes were overexpressed in a polycistronic manner; the coding gene for CkDdc was recombined into pCDFDuet-1 to obtain the recombinant plasmid pCDF- ckddc The recombinant plasmid pET30a- xsp4h_m -cv pcd - ecfolm and pCDF- ckddc Transfer to BL21 (Δ) tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL The engineered strain TRP-2 was obtained.

[0008] Furthermore, the encoding genes of CvPcd, EcFolM, and CkDdc are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0009] Furthermore, the method for constructing the high-L-tryptophan-producing Escherichia coli strain BL21 (ΔtnaA / ΔtrpR / ΔTyrA / ΔpheA / ΔTrpL) 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 ).

[0010] Based on the above, the present invention provides a method for synthesizing serotonin, using glucose as raw material and lactose or IPTG as an inducer, and using the above-mentioned recombinant engineered strain TRP-2 to ferment and synthesize serotonin, namely 5-hydroxytryptamine.

[0011] 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 0.1-1 g / L.

[0012] The aforementioned recombinant engineered strain TRP-2 can be used in the biosynthesis of serotonin.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This 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: a mutant of phenylalanine hydroxylase (XsP4H_m), dehydratase (CvPcd), reductase (EcFolM), and decarboxylase (CkDdc). The tryptophan derivative serotonin was synthesized by bio-fermentation, and the efficient biosynthesis of serotonin could be achieved at room temperature and normal pressure. Attached Figure Description

[0014] Figure 1 This describes the biosynthetic route of melatonin.

[0015] Figure 2 HPLC analysis of the fermentation broth of engineered strain TRP-1.

[0016] Figure 3 HPLC analysis of the fermentation broth of engineered strain TRP-2.

[0017] Figure 4 HPLC analysis of the fermentation broth of engineered strain TRP-3. Detailed Implementation

[0018] 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.

[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 / ΔtrpR / 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 Using the fragment as a template, overlap extension PCR was performed using primers F1 / R3 to amplify the fusion fragment. xsp4h - cvpcd - ecfolm The target fragment was recovered using a gel extraction kit, and the pET30a plasmid was double-digested with BamHI and XhoI restriction endonucleases, respectively. xsp4h - cvpcd - ecfolmThe fusion fragment was recovered using a gel extraction kit. T4 ligase was then used to ligate the fragment. xsp4h - cvpcd - ecfolm The fragment was linked to the linearized vector pET30a to construct pET30a- xsp4h - cvpcd - ecfolm Recombinant plasmid. The enzyme-linked product was then heat-shocked and transformed into *E. coli* DH5α competent cells, and subsequently plated on LB agar plates containing 50 mg / L kanamycin. Single colonies from the selection plate were picked, cultured in 25 mL of LB broth containing kanamycin, and then plasmid was extracted. Sequencing analysis confirmed the sequence was correct, and the plasmid was considered recombinant pET30a-. xscp4h - cvpcd - ecfolm .

[0023] With 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℃, 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 were then spread onto LB plates containing 50 mg / L kanamycin resistance.

[0024] After successful sequencing confirmation of the mutation, the plasmid was extracted and used as a template for a new round of site-directed mutagenesis, finally obtaining the recombinant plasmid pET30a- carrying the XsP4H three-point mutant XsP4H-W229F / L148I / A179K (XsP4H_m). xsp4h_m - cvpcd - ecfolm .

[0025] Using the same method, recombinant plasmid pET30a- was constructed. xsp4h_m - ckddc - cvpcd - ecfolm pCDF- ckddc and pCDF- sgaanat - hsasmt .

[0026] Table 1. Primer Information

[0027] Example 2: Genome Editing of Chassis Cells by tnaA Taking the knockout of a cell as an example, this paper introduces the process of editing the chassis cell genome.

[0028] 1) Targeting plasmid pTarget F- tnaA Construction of -gRNA Using plasmid pTarget F as a template, primer pairs were used... tnaA -F / tnaA -R (Table 1), perform full plasmid circular PCR to construct the target plasmid pTarget F- tnaA -gRNA. The obtained PCR product was digested with DMT (37℃, 1 h) and then heat-shocked to transform into *E. coli* DH5α competent cells. Transformants were picked for culture and plasmid extraction, and DNA sequencing confirmed the targeting plasmid pTarget F- tnaA -gRNA was successfully constructed.

[0029] 2) Amplification of repair template by E. coil Using BL21(DE3) bacterial culture as a template, primer pairs were used... tnaA -UF / tnaA -UR and tnaA -DF / tnaA -DR (Table 1) amplification tnaA The upstream and downstream homologous arms of the gene. After purification and recovery, using the two fragments as templates, primer pairs were used... tnaA -UF / tnaA -DR was used to amplify the fusion fragment of the upstream and downstream homologous arms by PCR, and after purification and recovery, a repair template of 1091 bp was obtained.

[0030] 3) Conversion and Screening The target plasmid pTarget F- was converted using an electroporation method. tnaA -gRNA and repair template were simultaneously transferred into a plasmid carrying pEcCas. E. coil BL21(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. The 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-reduced DNA was then spread 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 identified as the plasmid-depleted gene-edited strain BL21 (Δ...). 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 Gene-edited strain BL21 (Δ) was transformed using a heat shock transformation method. tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpLThe cells were collected from competent cells and then spread onto LB agar plates containing 50 mg / L kanamycin. The cells were incubated at 37°C for 12-16 h until single colonies appeared, which were then identified as the engineered strain BL21 (Δ) producing high levels of 5-hydroxytryptophan. tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL pET30a- xsp4h_m - cvpcd - ecfolm It was named TRP-1.

[0035] Tests showed that the shake-flask fermentation yield of strain TRP-1 was significantly higher than that of the wild-type high-yield 5-hydroxytryptophan-producing engineered strain BL21 (Δ). tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL pET30a- xsp4h - cvpcd - ecfolm Increased by 68% (0.52 g / L vs. 0.31 g / L).

[0036] Example 4: Fermentation Synthesis of 5-HTP 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 (containing 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... 600When 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.

[0037] Example 5: Construction of engineered bacteria producing high levels of 5-HT The recombinant plasmid pET30a- xsp4h_m - cvpcd - ecfolm and pCDF- ckddc Gene-edited strain BL21 (Δ) was introduced via heat shock co-transformation. tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL The cells were collected as competent cells and then spread onto LB agar plates containing 50 mg / L kanamycin and 50 mg / L streptomycin. The cells were incubated at 37°C for 12–16 h until a single colony appeared, which was the high-5-HT-producing engineered strain BL21 (Δ...). tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL pET30a- xsp4h_m - cvpcd - ecfolm pCDF- ckddc It was named TRP-2.

[0038] Example 6: Fermentation Synthesis of 5-HT 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 of the engineered strain 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 loading 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 of 5-HT was obtained.

[0039] Example 7: Construction of melatonin-producing engineered bacteria Recombinant plasmid pET30a- xsp4h_m - ckddc - cvpcd - ecfolm and pCDF- sgaanat - hsasmt Gene-edited strain BL21 (Δ) was introduced via heat shock co-transformation. tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL The cells were harvested from competent cells and then spread onto LB agar plates containing 50 mg / L kanamycin and 50 mg / L streptomycin. The cells were incubated at 37°C for 12-16 h until single colonies appeared, which was then identified as the high-melatonin-producing engineered strain BL21 (Δ...). tnaA / Δ trpR / Δ TyrA / Δ pheA / Δ TrpL pET30a- xsp4h_m - ckddc - cvpcd - ecfolm pCDF- sgaanat - hsasmtIt was named TRP-3.

[0040] Example 8: Fermentation Synthesis of Melatonin 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 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. 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.

[0041] In summary, the engineered strains constructed in this invention exhibited high production capacity during batch feed fermentation in a 5 L fermenter. The engineered strain TRP-1, fermented at 25°C for 40 h, synthesized 26.9 g / L of 5-HTP, representing the highest yield of 5-HTP synthesized using biological methods to date. The engineered strain TRP-2, fermented at 25°C for 40 h, synthesized 8.9 g / L of 5-HT without producing the byproduct tryptamine (TRPM). The engineered strain TRP-3, fermented at 30°C for 60 h, synthesized 2.1 g / L of melatonin.

[0042] 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 recombinant engineered strain TRP-2, characterized in that, The recombinant engineered strain TRP-2 was obtained by transforming a high-L-tryptophan-producing Escherichia coli strain into an expression vector containing a phenylalanine hydroxylase mutant, CvPcd, EcFolM, and CkDdc encoding genes. 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-2 according to claim 1, characterized in that, The encoding genes for CvPcd, EcFolM, and CkDdc are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

3. The recombinant engineered strain TRP-2 according to claim 1, characterized in that, The method for constructing the Escherichia coli strain that produces high levels of L-tryptophan 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 serotonin, characterized in that, Serotonin was synthesized by fermentation using glucose as raw material and lactose or IPTG as an inducer, based on the recombinant engineered strain TRP-2 as described in any one of claims 1 to 3.

5. The method for synthesizing serotonin 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-2 as described in any one of claims 1 to 3 in serotonin synthesis.

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