Genetically engineered bacterium for synthesizing melatonin and application of genetically engineered bacterium

Abstract

The invention relates to the field of genetic engineering and fermentation engineering, and discloses a genetically engineered bacterium for producing melatonin and application of the genetically engineered bacterium in fermentation production of melatonin. According to the invention, genes of mtrA, PTPS, SPR, PCD and DHPR of related enzymes synthesized by coding 2-amino-6-(1, 2-dihydroxypropyl)-5, 6, 7, 8-tetrahydro-4 (1H)-pterin diketone and genes of L-2-amino-3 (beta-indole) propionic acid monooxygenase, oxymethyltransferase, N-acetyltransferase and decarboxylase are co-expressed on the genetically engineered bacterium, so that the gene engineering bacterium is obtained. The strain can be used for fermentation production of melatonin, and experiments show that the yield of the strain can reach 6480 mg / L.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and fermentation engineering, and specifically to the construction of a melatonin-synthesizing genetically engineered bacterium and its application in the fermentation preparation of melatonin. Background Technology

[0002] Melatonin (MT) is a peptide hormone primarily secreted by the pineal gland that has broad regulatory effects on many systems. For example, in the digestive system, melatonin protects the gastric mucosa, increases gastric blood flow, and can inhibit the occurrence and development of ulcers. Exogenous melatonin is considered a health supplement in my country and is mainly used to regulate mild insomnia, reduce sleep latency, and increase sleep duration.

[0003] Currently, melatonin production is primarily based on chemical synthesis. However, chemical synthesis of melatonin suffers from problems such as cumbersome steps, low yield, and non-unique chirality. Furthermore, chemical synthesis is characterized by high energy consumption and pollution. Compared to chemical synthesis, biosynthesis offers advantages such as environmental friendliness, low energy consumption, and green environmental protection. Although the biosynthetic pathway of melatonin is well understood, the proteins involved have low specific activity, making them difficult to express in existing mature expression systems. They often form inclusion bodies or exhibit extremely low expression levels. Currently, there are no engineered bacteria capable of producing high levels of melatonin. Therefore, it is necessary to develop a high-yield melatonin strain to achieve efficient, large-scale industrial production of melatonin via fermentation. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies, such as low melatonin yield and poor enzyme element performance, and to provide a genetically engineered bacterium that can directly generate melatonin from tryptophan by fermentation and its application in the fermentation production of melatonin. Using the genetically engineered bacterium of this invention, high-yield melatonin production can be achieved.

[0005] Through prior research, the inventors of this invention constructed a system co-expressing L-2-amino-3(β-indole)propionate monooxygenase, cyclase mtrA, 6-pyruvyltetrahydrobiopterin synthase PTPS, guanopterin reductase SPR, and pterin-4α-

[0006] The genes for methanolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) were used to engineer a bacterium that synthesizes 5-hydroxytryptophan from tryptophan. Based on this, oxygen methyltransferase (COMT), N-acetyltransferase (AANAT), and decarboxylase (TDC) were introduced to construct an engineered bacterium that synthesizes melatonin from tryptophan in a one-step fermentation process.

[0007] This invention provides a genetically engineered bacterium for producing melatonin, which is obtained by introducing genes encoding oxymethyltransferase and / or N-acetyltransferase and decarboxylase into Escherichia coli for co-expression via molecular cloning.

[0008] Preferably, it also contains genes encoding cyclization hydrolase mtrA, 6-pyruvyltetrahydrobiopterin synthase PTPS, guanopterin reductase SPR, pterin-4α-methanolamine dehydratase PCD and dihydropteridine reductase DHPR, as well as L-2-amino-3(β-indole)propionate monooxygenase, oxymethyltransferase (COMT), N-acetyltransferase (AANAT) and decarboxylase (TDC).

[0009] Particularly preferred is the Escherichia coli strain BL21(DE3).

[0010] More preferably, the originating strain, *Escherichia coli*, does not express one and / or two and / or three and / or four genes from the following: tryptophanase tnaA, tryptophan operon repressor protein trpR, aromatic amino acid transporter yddG, and S-adenosylmethionine decarboxylase speD. Specifically, this can be achieved by knocking out these genes using genetic engineering techniques such as gene editing.

[0011] This invention provides the application of the genetically engineered bacteria in the preparation of melatonin.

[0012] The present invention provides a method for preparing melatonin, comprising the following steps: fermenting and culturing the genetically engineered bacteria to produce melatonin, optionally further comprising the step of isolating the melatonin.

[0013] Preferably, a feeding method is used during the fermentation process. Preferably, the fermentation time is 20-60 hours, more preferably 30-50 hours, such as 40-48 hours.

[0014] Specifically, the fermentation medium used for fermentation culture has the following formula: 5 g / L-10 g / L glycerol, 5 g / L-10 g / L diammonium hydrogen phosphate, 10.5 g / L potassium dihydrogen phosphate, 3.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid, 5 mL 200× trace elements, ammonia water to adjust pH to 7.2, 0.1% defoamer, and tryptophan 1 g / L-10 g / L; among which, the 200× trace elements are: 20 g / L ferrous sulfate heptahydrate, 4 g / L calcium chloride, 4.4 g / L zinc sulfate heptahydrate, 1 g / L manganese sulfate tetrahydrate, 2 g / L copper sulfate pentahydrate, 0.2 g / L hexaammonium molybdate tetrahydrate, and 0.04 g / L sodium borate decahydrate.

[0015] The feeding medium in the feeding method consists of 200g / L-600g / L glycerol, 1g / L-10g / L peptone, and 1g / L-20g / L yeast extract.

[0016] Preferably, the fermentation medium is formulated as follows: 10 g / L glycerol, 6 g / L diammonium hydrogen phosphate, 10.5 g / L potassium dihydrogen phosphate, 3.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid, 5 mL 200× trace elements, ammonia water to adjust pH to 7.2, 0.1% defoamer, and 10 g / L tryptophan.

[0017] Feeding medium: 600 g / L glycerol, 10 g / L peptone, 20 g / L yeast extract.

[0018] Specifically, the fermentation temperature is 35-39℃ and the rotation speed is 180-250 rpm; for fermenter culture, the rotation speed is 500-900 rpm and the maximum aeration rate is 7 L / min.

[0019] The genetically engineered bacteria constructed in this invention can ferment and produce melatonin, with a maximum yield of 6480 mg / L. Therefore, this invention provides a novel biosynthetic pathway for high-yield melatonin production, laying the foundation for large-scale industrial production of melatonin and possessing significant scientific research value and social benefits. Attached Figure Description

[0020] Figure 1 The results are from a 5L fermentation tank.

[0021] Figure 2 The results are from the liquid phase analysis of the fermentation broth. Detailed Implementation

[0022] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation of the present invention.

[0023] Example 1: Construction of a strain co-expressing melatonin synthase

[0024] 1. The vector pET24a-YC100 described in Chinese patent application 202310237287.9 ​​is used to co-express COMT derived from Arabidopsis thaliana (NCBI: NC_003076.8) before or after the expression of C5-monooxygenase (AAD01923.1) to obtain co-expression vectors pET24a-MT1 and pET24a-MT2. Specifically, pET24a-MT1 is expressed in tandem before C5-monooxygenase, and pET24a-MT2 is expressed in tandem after C5-monooxygenase. Further co-expression of pET24a-MT1 derived from Harmonia thaliana is also possible. The TDCs of axyridis (NCBI: AMQ13055.1) were used to obtain co-expression vectors pET24a-MT3, pET24a-MT4, and pET24a-MT5. The expression of pET24a-MT3 is achieved by tandem TDCs after pET24a-MT1; pET24a-MT4 is achieved by tandem TDCs after pET24a-MT2; and pET24a-MT5 is achieved by tandem TDCs in the middle of pET24a-MT1.

[0025] 2. Similarly, the construct pACYC-Duet-YC101 in Chinese patent application 202310237287.9, based on the expression of mtrA (WP_038828581.1), PTPS (NP_000308.1), SPR (NP_003115.1), PCD (WP_004659699.1), and DHPR (WP_000351487.1), co-expresses AANAT from patent (application number 2024100254054) and Harmonia based on the expression of mtrA (WP_038828581.1), PTPS (NP_000308.1), SPR (NP_003115.1), PCD (WP_004659699.1), and DHPR (WP_000351487.1). The TDC of axyridis (NCBI: AMQ13055.1) yielded co-expression vectors pACYC-Duet-MT1, pACYC-Duet-MT2, and pACYC-Duet-MT3. Among them, pACYC-Duet-MT1 is formed by tandem AANAT before the TDC; pACYC-Duet-MT2 is formed by tandem AANAT after the TDC; and pACYC-Duet-MT3 is formed by tandem expression of AANAT only on pACYC-Duet-YC101.

[0026] 3. The pACYC-Duet sequence vector and the pET24a sequence vector were co-transformed into Escherichia coli BL21(DE3) to obtain six genetically engineered bacteria (A, B, C, D, E, and F) (see Table 1 for the corresponding vectors introduced). Single colonies were picked and inoculated into 5 mL of LB medium (containing a final concentration of 50 μg / mL Kan and 5 μg / mL Cm) and incubated overnight at 37°C and 220 rpm.

[0027] 4. Inoculate 50 mL of TB medium (12 g / L peptone, 24 g / L yeast extract, 8 mL / L glycerol, 2.31 g KH2PO4, 16.43 g K2HPO4) at a 5% inoculation rate, and add Kan and Cm to a final concentration of 50 μg / mL. Incubate at 37℃ and 220 rpm until the OD600 reaches approximately 1.0, then add IPTG to a final concentration of 0.1 mM and incubate at 25℃ for 12 h to induce protein expression.

[0028] 5. 4 g / L L-tryptophan and 6 g / L methionine were directly added to a 50 mL system for shake-flask fermentation. Samples were taken after 36 hours to detect melatonin (MT) production. Liquid chromatography analysis was performed, and the results are as follows:

[0029] Table 1. Fermentation results of different expression vectors

[0030] strain expression carrier MT (g / L) A pACYC-Duet-MT1&pET24a-MT1 1.0 B pACYC-Duet-MT2&pET24a-MT1 1.2 C pACYC-Duet-MT1&pET24a-MT2 1.3 D pACYC-Duet-MT3&pET24a-MT3 1.5 E pACYC-Duet-MT3&pET24a-MT4 1.8 F pACYC-Duet-MT3&pET24a-MT5 1.2

[0031] Example 2: Construction of melatonin synthesis chassis cells and melatonin-producing strains

[0032] Using a dual plasmid knockout system, genes ΔtnaA, ΔtrpR, ΔyddG, and ΔspeD were knocked out separately via homologous arms to obtain chassis cells BL21(DE3)ΔtnaA, BL21(DE3)ΔtnaAΔtrpR, BL21(DE3)ΔtnaAΔyddG, BL21(DE3)ΔtnaAΔtrpRΔyddG, and BL21(DE3)ΔtnaAΔtrpRΔyddGΔspeD.

[0033] Melatonin-producing strains were obtained by co-transforming pET24a-MT4 and pACYC-Duet-MT3 into BL21(DE3), BL21(DE3)ΔtnaA, BL21(DE3)ΔtnaAΔtrpR, BL21(DE3)ΔtnaAΔyddG, BL21(DE3)ΔtnaAΔtrpRΔyddG, and BL21(DE3)ΔtnaAΔtrpRΔyddGΔspeD, respectively, and were designated as 1: BL21(DE3), 2: BL21(DE3)ΔtnaA, 3: BL21(DE3)ΔtnaAΔtrpR, 4: BL21(DE3)ΔtnaAΔyddG, 5: BL21(DE3)ΔtnaAΔtrpRΔyddG, and 6: BL21(DE3)ΔtnaAΔtrpRΔyddGΔspeD.

[0034] Example 3: Synthesis of melatonin by shake-flask fermentation

[0035] 1. The six types of chassis cells obtained in Example 2 were electroporated to prepare competent cells, and melatonin synthesis-related plasmids were electroporated into different chassis cells.

[0036] 2. Pick a single colony of the successfully constructed strain and inoculate it into 5 mL of LB medium (containing a final concentration of 50 μg / mL Kan and 5 μg / mL Cm), and incubate overnight at 37℃ and 220 rpm.

[0037] 3. Inoculate 50 mL of TB medium (12 g / L peptone, 24 g / L yeast extract, 8 mL / L glycerol, 2.31 g KH2PO4, 16.43 g K2HPO4) at a 5% inoculation rate, and add Kan and Cm to a final concentration of 50 μg / mL. Incubate at 37℃ and 220 rpm until the OD600 reaches approximately 1.0, then add IPTG to a final concentration of 0.1 mM and incubate at 25℃ for 12 h to induce protein expression.

[0038] 4. Add 4 g / L of L-tryptophan and 6 g / L of methionine directly to a 50 mL system, carry out shake-flask fermentation, and take samples to detect melatonin production.

[0039] 5. The liquid phase detection results are shown in Table 2. After 36 hours of shake-flask fermentation, the chassis cells BL21(DE3)ΔtnaAΔtrpRΔyddGΔspeD can produce 2.03 g / L of melatonin.

[0040] Table 2. Results of cell fermentation in different chassis

[0041]

[0042] Example 4: Melatonin synthesis via fermentation in a 5L tank

[0043] 1. Select production strain 6: BL21(DE3)ΔtnaAΔtrpRΔyddGΔspeD and inoculate a single colony into a 5 mL test tube of culture medium (containing a final concentration of 50 μg / mL Kan and 5 μg / mL Cm), and incubate overnight at 37℃ and 220 rpm.

[0044] 2. Inoculate 1% of the culture medium into 100 mL of LB medium and incubate overnight at 37°C and 220 rpm.

[0045] 3. Inoculate 10% of the inoculum into a 5L fermenter (containing 2L of fermentation broth), rotate at 500-900 rpm, with a maximum aeration rate of 7L / min and a temperature of 37℃.

[0046] 4. Cultivating OD 600Around 20°C, add IPTG to a final concentration of 0.1M, induce protein expression at 25°C for 12 hours, add 6g / L methionine, and continue fermentation at 30°C. Take samples at time points to detect the production of melatonin, and control the pH to 7.0 with ammonia water (25% ammonia water).

[0047] 5. Fermentation medium formulation: 10 g / L glycerol, 6 g / L diammonium hydrogen phosphate, 10.5 g / L potassium dihydrogen phosphate, 3.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid, 5 mL 200× micronutrient solution, pH adjusted to 7.2 with ammonia, 0.1% antifoaming agent, 10 g / L tryptophan. 200× micronutrient solution: 20 g / L ferrous sulfate heptahydrate, 4 g / L calcium chloride, 4.4 g / L zinc sulfate heptahydrate, 1 g / L manganese sulfate tetrahydrate, 2 g / L copper sulfate pentahydrate, 0.2 g / L hexaammonium molybdate tetrahydrate, 0.04 g / L sodium borate decahydrate. Fed medium: 600 g / L glycerol, 10 g / L peptone, 20 g / L yeast extract.

[0048] 6. Take 200 μL of fermentation broth, add 800 μL of 50% methanol and 0.1% trifluoroacetic acid solution, shake and centrifuge, then pass through a membrane and perform liquid chromatography to detect the amounts of melatonin and tryptophan.

[0049] 7. The liquid chromatography detection conditions were as follows: column: Zorbax SB-C18, StableBond Analytical 4.6X150mm, detection wavelength: 275nm, column temperature: 35℃, flow rate: 1mL / min, sample loading volume: 10μL, detection time: 10min, mobile phase A: pure water (containing 0.1% trifluoroacetic acid), mobile phase B: pure methanol, mobile phase A: mobile phase B = 60:40.

[0050] 8. Liquid chromatography analysis revealed the presence of tryptophan (Trp) and melatonin (MT).

[0051] The results showed that the genetically engineered bacterial strain 6: BL21(DE3)ΔtnaAΔtrpRΔyddGΔspeD could produce melatonin in a 5L fermenter after 20 hours, and after 44 hours of fermentation, the melatonin yield reached a maximum of 6480 mg / L. The results are shown in Table 3. Figure 1 and Figure 2 As shown.

[0052] Table 3. Results of melatonin production from fermentation in a 5L tank.

[0053] Time (h) <![CDATA[OD 600 ]]> Trp(g / L) MT (g / L) 0 1 9.44 0 4 3 9.44 0 8 11 9.44 0 10 20 9.15 0 20 65 7.64 0.84 22 74 7 1.59 24 80 6.45 2.72 28 102 5.57 4.77 32 118 4.93 5.60 44 120 3.43 6.48 48 118 2.99 6.31 52 110 2.84 6.08 .

Claims

1. A genetically engineered bacterium that produces melatonin, characterized in that, The genetically engineered bacteria are obtained by introducing genes encoding oxymethyltransferase and / or N-acetyltransferase and decarboxylase into Escherichia coli for co-expression via molecular cloning.

2. The genetically engineered bacterium according to claim 1, characterized in that, It also contains genes encoding cyclase, 6-pyruvyltetrahydropterin synthase, guanopterin reductase, pterin-4α-methanolamine dehydratase, dihydropteridine reductase, as well as L-2-amino-3(β-indole)propionate monooxygenase, oxymethyltransferase, N-acetyltransferase, and decarboxylase related to the synthesis of 2-amino-6-(1,2-dihydroxypropyl)-5,6,7,8-tetrahydro-4(1H)-pterindione, and L-2-amino-3(β-indole)propionate monooxygenase, oxymethyltransferase, N-acetyltransferase, and decarboxylase.

3. The genetically engineered bacteria according to claim 2, characterized in that, The Escherichia coli mentioned is Escherichia coli strain BL21(DE3).

4. The genetically engineered bacteria according to claim 3, characterized in that, The originating bacteria, *Escherichia coli*, does not express tryptophanase. tnaA、 Tryptophan operon repressor protein trpR, Aromatic amino acid transporters yddG、 S-adenosylmethionine decarboxylase speD One and / or two and / or three and / or four genes in it.

5. The use of the genetically engineered bacteria as described in any one of claims 1 to 4 in the preparation of melatonin.

6. A method for preparing melatonin, characterized in that, The method includes the following steps: fermenting and culturing the genetically engineered bacteria as described in any one of claims 1 to 4 to produce melatonin, optionally further including the step of isolating the melatonin.

7. The method as described in claim 6, characterized in that, The fermentation process is carried out by feeding. Preferably, the fermentation time is 20-60 hours, more preferably 30-50 hours, such as 40-48 hours.

8. The method as described in claim 7, characterized in that, The fermentation medium used for fermentation culture has the following formula: 5 g / L-10 g / L glycerol, 5 g / L-10 g / L diammonium hydrogen phosphate, 10.5 g / L potassium dihydrogen phosphate, 3.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid, 5 mL 200× trace elements, pH adjusted to 7.2 with ammonia water, 0.1% defoamer, and 1 g / L-10 g / L tryptophan; among which, the 200× trace elements consist of: 20 g / L ferrous sulfate heptahydrate, 4 g / L calcium chloride, 4.4 g / L zinc sulfate heptahydrate, 1 g / L manganese sulfate tetrahydrate, 2 g / L copper sulfate pentahydrate, 0.2 g / L hexaammonium molybdate tetrahydrate, and 0.04 g / L sodium borate decahydrate. The feeding medium in the feeding method consists of 200 g / L-600 g / L glycerol, 1 g / L-10 g / L peptone, and 1 g / L-20 g / L yeast extract.

9. The method according to claim 8, characterized in that, The fermentation medium formula is as follows: 10 g / L glycerol, 6 g / L diammonium hydrogen phosphate, 10.5 g / L potassium dihydrogen phosphate, 3.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid, 5 mL 200× trace elements, ammonia water to adjust pH to 7.2, 0.1% antifoaming agent, and 10 g / L tryptophan. Feeding medium: 600 g / L glycerol, 10 g / L peptone, 20 g / L yeast extract.

10. The method according to claim 6, characterized in that, The fermentation temperature is 35-39℃ and the rotation speed is 180-250 rpm; for fermenter culture, the rotation speed is 500 rpm-900 rpm and the maximum aeration rate is 7 L / min.

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