Yeast engineering strain for synthesizing melatonin as well as construction method and application of yeast engineering strain

Abstract

The invention provides a yeast engineering strain for synthesizing melatonin as well as a construction method and application of the yeast engineering strain, and relates to the technical field of genetic engineering and microbial fermentation engineering. According to the invention, a novel yeast engineering bacterium for synthesizing melatonin is constructed, wherein the novel yeast engineering bacterium is used for overexpressing arylalkylamine N-acetyl transferase, 5-hydroxytryptophan decarboxylase, tryptophan hydroxylase, N-acetyl serotonin methyltransferase, 6-pyruvoyl tetrahydrobiopterin synthetase, ink pterin reductase, 4a-hydroxytetrahydrobiopterin dehydratase and dihydropterin reductase. Under the fermentation process that 1g / L tryptophan is externally added, the content of melatonin in a fermentation product obtained by the yeast engineering bacteria reaches 14.32 mg / L, and 17.48 mg / g DCW. Under the condition that 4g / L tryptophan is added, 5L fermentation tank culture is carried out, and the content of melatonin in a fermentation product obtained by the yeast engineering bacteria reaches 91.82 mg / L. The engineered yeast provided by the invention can significantly increase the yield of melatonin, is expected to realize industrial mass production of melatonin, and has good application value and commercial value.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and microbial fermentation engineering, and in particular to an engineering yeast strain for synthesizing melatonin, a construction method and application thereof. Background Art

[0002] Melatonin is a natural mammalian hormone that plays an important role in regulating the human circadian rhythm. It also has multiple physiological functions such as antioxidants, antibacterial and anti-inflammatory effects. It is widely used in many fields such as food, chemical industry, pharmaceuticals and agriculture, and has a broad market demand. The global market size of melatonin in 2023 is US$1.736 billion, and it is expected to reach US$3.93 billion by 2028. At present, the technologies for industrial production of melatonin mainly include chemical synthesis and plant and animal extraction. Due to the reduction of petroleum resources and the increasingly serious environmental pollution, the disadvantages of traditional synthesis methods are becoming increasingly apparent, with limitations such as high cost, long synthesis time, and cumbersome process. Therefore, in recent years, the use of microbial cell factories to produce melatonin from renewable resources has received increasing attention.

[0003] At present, melatonin fermentation strains are mainly divided into two categories. The first category is natural melatonin-producing strains, such as Saccharomyces cerevisiae and Pseudomonas fluorescens, and the second category is engineered bacteria modified by metabolic engineering, mainly including Escherichia coli and Saccharomyces cerevisiae. Among them, although natural melatonin-producing strains can be used to produce melatonin, their shortcomings are also very obvious, such as low fermentation production efficiency and high culture costs. Because yeast grows well, has excellent and stable performance, and has significant resistance to stress under various industrial conditions, the use of yeast engineered bacteria to ferment melatonin has become a research hotspot. It is necessary to conduct further in-depth research on yeast to lay the foundation for the commercial production of melatonin.

[0004] Studies have shown that the melatonin synthesis pathway is relatively well studied in animals and plants, mainly using tryptophan as a precursor, and finally generating melatonin through four steps of enzymatic reactions. Due to the complexity of the microbial metabolic network and the diversity of secondary metabolites, the melatonin synthesis pathway of many species has not been studied. Therefore, microbial synthesis of melatonin mainly involves screening various putative melatonin synthesis genes from animals and plants, and constructing heterologous expression systems to promote the mass production of melatonin.

[0005] At present, the construction of synthetic melatonin engineering bacteria such as Saccharomyces cerevisiae and Escherichia coli has been realized through transgenic technology and gene editing technology. For example: the invention patent with publication number CN 111394269 A discloses that the maximum melatonin production of Saccharomyces cerevisiae reaches 25.7 mg / L by accumulating the precursor substance tryptophan. The invention patent with publication number CN 112899176A constructs a Saccharomyces cerevisiae engineering strain with a production of 35 mg / L by overexpressing acetyl complex amine-O-methyltransferase ASMT1. The invention patent with publication number CN 115806891 A constructs a high-yield melatonin synthesis strain by introducing human-derived tryptophan hydroxylase, aromatic amino acid decarboxylase, tryptophan hydroxylase and aromatic amino acid decarboxylase. Under the fermentation process conditions of supplementing tryptophan, the melatonin production can reach 860 mg / L. In addition, in the melatonin synthesis pathway, aromatic amino acid hydroxylase requires tetrahydrobiopterin (BH4) as a cofactor to improve its catalytic efficiency. However, directly adding tetrahydrobiopterin during the fermentation process can easily lead to high culture costs. Therefore, it is necessary to explore the synthesis pathways involved in tetrahydrobiopterin to further promote the large-scale production of melatonin by microorganisms. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a yeast engineering strain for synthesizing melatonin based on the tetrahydrobiopterin pathway and a construction method and application thereof.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] In a first aspect, the present invention provides a yeast engineered bacterium for synthesizing melatonin, wherein the yeast engineered bacterium is constructed by a starting strain and genes of enzymes related to melatonin biosynthesis, wherein the starting strain is Saccharomyces cerevisiae; the enzymes related to melatonin biosynthesis include at least one of 5-hydroxytryptophan decarboxylase (5-hydroxy-L-tryptophan decarboxylase, DDC), arylalkylamine N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT), N-acetylserotonin methyltransferase (N-acetylserotoninmethyltransferase, AMST) / hydroxyindole O-methyltransferase (Hydroxyindole-O-methyltransferase, HIOMT) and tryptophan 5-hydroxylase (tryptophan5-hydroxylase, TPH).

[0011] Furthermore, the melatonin biosynthesis-related enzymes also include at least one of 6-pyruvoyl-tetrahydropterin synthase (GgPTS), sepiapterin reductase (GgSPR), 4a-hydroxytetrahydrobiopterin dehydratase (GgPCBD1), and quinoid dihydropteridine reductase (GgQDPR).

[0012] Among the above-mentioned melatonin biosynthesis-related enzymes, the N-acetylserotonin methyltransferase / hydroxyindole O-methyltransferase is derived from, including but not limited to, Arabidopsis thaliana, Malus domestica, Oryza sativaL, Manihot esculenta and Homo sapiens; the arylalkylamine N-acetyltransferase is derived from Gallus gallus; the 5-hydroxytryptophan decarboxylase is derived from Homo sapiens; the tryptophan hydroxylase is derived from Gallus gallus; the 6-pyruvoyltetrahydrobiopterin synthase is derived from Gallus gallus; the septopterin reductase is derived from Gallus gallus; the dihydropteridine reductase is derived from Gallus gallus; and the 4a-hydroxytetrahydrobiopterin dehydratase is derived from Gallus gallus.

[0013] In a second aspect, the present invention provides a method for constructing an engineered yeast bacterium for synthesizing melatonin, wherein the construction method comprises: introducing a gene encoding an enzyme related to melatonin biosynthesis into a cerevisiae strain and overexpressing the enzyme.

[0014] Among the genes encoding the above-mentioned melatonin biosynthesis-related enzymes, the N-acetylserotonin methyltransferase / hydroxyindole O-methyltransferase is shown in SEQ ID NO.1-5; the nucleotide sequence of the arylalkylamine N-acetyltransferase encoding gene is shown in SEQ ID NO.6; the nucleotide sequence of the 5-hydroxytryptophan decarboxylase encoding gene is shown in SEQ ID NO.7; the nucleotide sequence of the tryptophan hydroxylase encoding gene is shown in SEQ ID NO.8; the nucleotide sequence of the 6-pyruvoyltetrahydrobiopterin synthase encoding gene is shown in SEQ ID NO.9; the nucleotide sequence of the septopterin reductase encoding gene is shown in SEQ ID NO.10; the nucleotide sequence of the dihydropteridine reductase encoding gene is shown in SEQ ID NO.11; the nucleotide sequence of the 4a-hydroxytetrahydrobiopterin dehydratase encoding gene is shown in SEQ ID NO.12.

[0015] In a third aspect, the present invention provides the use of the yeast engineering bacteria in the biosynthesis of melatonin, wherein the yeast engineering bacteria strain is fermented and cultured, and melatonin is isolated and obtained.

[0016] In a fourth aspect, the present invention provides the use of the above-mentioned yeast engineering bacteria in the preparation of melatonin-rich products, including foods, feed additives, health products, and medicines.

[0017] (III) Beneficial effects

[0018] The present invention first constructs a yeast engineering bacterium that overexpresses arylalkylamine N-acetyltransferase, 5-hydroxytryptophan decarboxylase, tryptophan hydroxylase, and N-acetylserotonin methyltransferase for synthesizing the basic pathway of melatonin. On this basis, in order to strengthen the synthesis of melatonin by yeast engineering bacteria, the above-mentioned strain is constructed and optimized for tetrahydrobiopterin synthesis and recycling pathways. The present invention constructs a novel yeast engineering bacterium for synthesizing melatonin that can also overexpress cofactors 6-pyruvyltetrahydrobiopterin synthase, septopterin reductase, 4a-hydroxytetrahydrobiopterin dehydratase, and dihydropterin reductase. Under the fermentation process of exogenously adding 1g / L tryptophan, the melatonin content in the fermentation product obtained by the yeast engineering bacteria reaches 14.32mg / L and 17.48mg / g DCW. Under the condition of adding 4g / L tryptophan, a 5L fermentation tank is cultured, and the melatonin content in the fermentation product obtained by the yeast engineering bacteria reaches 91.82mg / L. The engineered yeast provided by the present invention can significantly increase the yield of melatonin, is expected to achieve industrial mass production of melatonin, and has good application value and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the effects of methyltransferases from five different animal and plant sources on the synthesis of melatonin by yeast engineered bacteria; A is the melatonin accumulation of strain MT01-05; B is the melatonin accumulation of strain MT02 under the addition of different doses of N-acetyl serotonin.

[0020] Figure 2 The melatonin accumulation of strain MT08 under the condition of adding different doses of tryptophan.

[0021] Figure 3 The melatonin accumulation of strain MT12 under the condition of adding different doses of tryptophan.

[0022] Figure 4 These are the results of the fermentation experiment using MT12 strain in a 5L fermenter. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] 1. The gene synthesis in the following examples was completed by GenScript Biotech (Nanjing) Co., Ltd.; the primer synthesis and sequencing in this example were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0025] 2. The experimental methods used in the following examples, including plasmid construction, enzyme digestion, preparation of competent cells, transformation, etc., are all conventional methods unless otherwise specified; the specific experimental conditions can be determined by simple experiments when necessary.

[0026] 3. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] 4. The Saccharomyces cerevisiae strain CEN.PK 2-1c (genotype MATa; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2) involved in the following examples.

[0028] 5. The genes involved in the following embodiments, including AtCOMT from Arabidopsis thaliana, MdASMT9 from Malus domestica, OsCOMT from Oryza sativa L, MeCOMT from Manihotesculenta, HsCOMT and HsDDC from Homo sapiens, GgAANAT, GgTPH, GgPTS, GgSPR, GgDQPR and GgPCBD1 from Gallus gallus, were all codon-optimized and synthesized.

[0029] 6. LB solid medium in the following examples: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 20 g / L agar powder.

[0030] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0031] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose.

[0032] YPD solid medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 2 wt% agar powder.

[0033] Yeast selection nutrient-deficient medium: 8 g / L SD nutrient-deficient medium (SD / -His, SD / -Ura SD / -His-Ura), 20 g / L glucose, adjusted to pH 6.0.

[0034] SD solid medium: SD liquid selective medium supplemented with 2 wt% agar powder.

[0035] Example 1

[0036] Amplification of the original gene and preparation of the target plasmid

[0037] (I) Preparation of target gene

[0038] 1. According to the nucleotide sequence of the hydroxyindole O-methyltransferase gene AtCOMT (Gene ID: 835504) from Arabidopsis thaliana provided by NCBI, after codon optimization, the nucleotide sequence of AtCOMT was obtained as shown in SEQ ID NO.1.

[0039] 2. According to the nucleotide sequence of the N-acetylserotonin methyltransferase gene MdASMT9 (Sequence ID: AIZ00789.1) from Malus domestica provided by NCBI, after codon optimization, the nucleotide sequence of MdASMT9 was obtained as shown in SEQ ID NO.2.

[0040] 3. According to the nucleotide sequence of the hydroxyindole O-methyltransferase gene OsCOMT (Gene ID: 4344702) from Oryza sativa L provided by NCBI, the nucleotide sequence of OsCOMT was obtained after codon optimization as shown in SEQ ID NO.3.

[0041] 4. According to the nucleotide sequence of the hydroxyindole O-methyltransferase gene MeCOMT (Sequence ID: KU361337.1) from Manihot esculenta provided by NCBI, after codon optimization, the nucleotide sequence of MeCOMT was obtained as shown in SEQ ID NO.4.

[0042] 5. According to the nucleotide sequence of the acetyl serotonin O-methyltransferase gene HsCOMT (Gene ID: 1312) from Homo sapiens provided by NCBI, after codon optimization, the nucleotide sequence of HsCOMT was obtained as shown in SEQ ID NO.5.

[0043] 6. According to the nucleotide sequence of arylalkylamine N-acetyltransferase GgAANAT (GeneID: 396066) from Gallus gallus provided by NCBI, after codon optimization, the nucleotide sequence of GgAANAT was obtained as shown in SEQ ID NO.6.

[0044] 7. According to the nucleotide sequence of the 5-hydroxytryptophan decarboxylase gene HsDDC (GeneID: 1644) from Homo sapiens provided by NCBI, after codon optimization, the nucleotide sequence of HsDDC was obtained as shown in SEQ ID NO.7.

[0045] 8. According to the nucleotide sequence of the tryptophan hydroxylase gene GgTPH (GeneID: 395799) from Gallus gallus provided by NCBI, after codon optimization, the nucleotide sequence of GgTPH was obtained as shown in SEQ ID NO.8.

[0046] 9. According to the nucleotide sequences of 6-pyruvoyltetrahydrobiopterin synthase gene GgPTS, septopterin reductase gene GgSPR, dihydropteridine reductase gene GgQDPR and 4a-hydroxytetrahydrobiopterin dehydratase gene GgPCBD1 from Gallus gallus (Gene ID: 374144; ID: 425255; Gene ID: 426335; Gene ID: 395729) provided by NCBI, after codon optimization, the nucleotide sequences of GgPTS, GgSPR, GgQDPR and GgPCBD1 were obtained as shown in SEQ ID NOs. 9-12.

[0047] (II) Preparation of target plasmid

[0048] 1 Construction of plasmids pJFE3-AtCOMT, pJFE3-MdASMT9, pJFE3-MeCOMT, pJFE3-OsCOMT, and pJFE3-HsCOMT

[0049] Primers were designed using the codon-optimized AtCOMT, MdASMT9, MeCOMT, OsCOMT and HsCOMT gene sequences and the expression plasmid pJFE3 sequence to obtain BamHI-AtCOMT-F (SEQ ID NO.13) / PstI-AtCOMT-R (SEQ ID NO.14), BamHI-MdASMT9-F (SEQ ID NO.15) / PstI-MdASMT9-R (SEQ ID NO.16), BamHI-MeCOMT-F (SEQ ID NO.17) / PstI-MeCOMT-R (SEQ ID NO.18), BamHI-OsCOMT-F (SEQ ID NO.19) / PstI-OsCOMT-R (SEQ ID NO.20), BamHI-HsCOMT-F (SEQ ID NO.21) / PstI-HsCOMT-R (SEQ ID NO.22). NO.22) primer sequence; use the above primers to perform PCR amplification to obtain an assembly fragment carrying the corresponding terminal homologous sequence.

[0050] pJFE3 was digested with BamH I and Pst I endonucleases and then recovered and purified. The above restriction fragments and PCR products were connected using the Gibson assembly cloning kit (New England Biolabs (NEB), England) to construct pJFE3-AtCOMT, pJFE3-MdASMT9, pJFE3-MeCOMT, pJFE3-OsCOMT, and pJFE3-HsCOMT plasmids.

[0051] 2 Construction of plasmid pJ-MdASMT9-GgNNANT

[0052] Primers were designed based on the codon-optimized GgNNANT gene sequence and the expression plasmid pJFE3-MdASMT9 sequence to obtain TEF1-GgNNANT-F (SEQ ID NO.23) / PGK1-GgNNANT-R (SEQ ID NO.24) primer sequences; PCR amplification was performed using the above primers to obtain an assembly fragment carrying the corresponding terminal homologous sequence.

[0053] pJFE3-MdASMT9 was digested with Sal I and BamH I endonucleases and then recovered and purified. The above restriction fragments and PCR products were connected using the Gibson assembly cloning kit (New England Biolabs (NEB), England) to complete the construction of the pJ-MdASMT9-GgNNANT plasmid.

[0054] 3 Construction of plasmid pJ-MdASMT9-GgNNANT-HsDDC

[0055] Primers were designed based on the codon-optimized HsDDC gene sequence and the expression plasmid pJ-MdASMT9-GgNNANT sequence to obtain the HFF2-HsDDC-F (SEQ ID NO.25) / PGK1-HsDDC-R (SEQ ID NO.26) primer sequence, and PCR amplification was performed using the above primers to obtain an assembly fragment carrying the corresponding terminal homologous sequence.

[0056] pJ-MdASMT9-GgNNANT was digested with Sac I and Spe I endonucleases and then recovered and purified. The above restriction fragments and PCR products were connected using the Gibson assembly cloning kit (New England Biolabs (NEB), England) to complete the construction of pJ-MdASMT9-GgNNANT-HsDDC plasmid.

[0057] 4 Construction of plasmid pJ-MdASMT9-GgNNANT-HsDDC-GgTPH

[0058] Primers were designed based on the codon-optimized GgTPH gene sequence and the expression plasmid pJ-GgASMT-GgNNANT-HsDDC sequence to obtain:

[0059] TPH3-GgTPH-F (SEQ ID NO.27) / tXPR2-ADH1-GgTPH-R (SEQ ID NO.28) primer sequence, PCR amplification using the above primers to obtain an assembly fragment carrying the corresponding terminal homologous sequence.

[0060] pJ-GgASMT-GgNNANT-HsDDC was digested with Nar I endonuclease and then recovered and purified. The above enzyme-cut fragments and PCR products were connected using the Gibson assembly cloning kit (New England Biolabs (NEB), England) to complete the construction of pJ-MdASMT9-GgNNANT-HsDDC-GgTPH plasmid.

[0061] 5 Construction of plasmid pIYC06-PTS

[0062] Primers were designed based on the codon-optimized GgPTS gene sequence and the expression plasmid pIYC06 sequence to obtain the BamHI-GgPTS-F (SEQ ID NO.29) / SalI-GgPTS-R (SEQ ID NO.30) primer sequence, and PCR amplification was performed using the above primers to obtain the assembly fragment carrying the corresponding terminal homologous sequence.

[0063] pIYC06 was digested with BamH I and Sal I endonucleases, and then recovered and purified. The above restriction fragments and PCR products were connected using the Gibson assembly cloning kit (New England Biolabs (NEB), England) to complete the construction of pIYC06-PTS plasmid.

[0064] 6 Construction of plasmid pIYC06-PTS-SPR

[0065] Primers were designed based on the SPR gene sequence after codon optimization and the expression plasmid pIYC06-TPS sequence to obtain the XbaI-SPR-F (SEQ ID NO.31) / PstI-SPR-R (SEQ ID NO.32) primer sequence, and PCR amplification was performed using the above primers to obtain the assembly fragment carrying the corresponding terminal homologous sequence.

[0066] pIYC06-TPS was purified after being reversed by Xba I and Pst I. The above restriction fragments and PCR products were connected by Gibson assembly to complete the construction of pIYC06-PTS-SPR plasmid.

[0067] 7 Construction of plasmid pIYC06-PTS-SPR-QDPR

[0068] Primers were designed based on the codon-optimized QDPR gene sequence and the expression plasmid pIYC06-TPS sequence to obtain

[0069] TEF1-QDPR-F (SEQ ID NO.33) / CYC1-QDPR (SEQ ID NO.34) primer sequences were used to obtain the assembled fragments carrying the corresponding terminal homologous sequences by PCR amplification.

[0070] pIYC06-PTS-SPR was purified after reverse PCR, and the above enzyme-cut fragments and PCR products were connected by Gibson assembly to complete the construction of pIYC06-PTS-SPR-QDPR plasmid.

[0071] 8 Construction of plasmid pIYC06-PTS-SPR-QDPR-PCBD1

[0072] Primers were designed based on the codon-optimized PCBD1 gene sequence and the expression plasmid pIYC06-PTS-SPR-QDPR sequence to obtain

[0073] The assembled fragments carrying the corresponding terminal homologous sequences were obtained by PCR amplification using NotI-PCBD1-F (SEQ ID NO.35) / SacI-PCBD1-R (SEQ ID NO.36) primers.

[0074] pIYC06-TPS-SPR-QDPR was purified after being reversed by Not I and Sac I. The above enzyme fragments and PCR products were connected by Gibson assembly to complete the construction of pIYC06-PTS-SPR-QDPR-PCBD1 plasmid.

[0075] Construction of an engineered yeast strain capable of synthesizing melatonin

[0076] 1. Using the Saccharomyces cerevisiae strain CEN.PK 2-1c (genotype MATa; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2) as the starting strain, pJ-MdASMT9-GgNNANT-HsDDC-GgTPH and pIYC06-PTS-SPR-QDPR-PCBD1 were transferred into the above starting strain by chemical transformation, and Ura and His were used as screening markers to construct an engineered strain MT12 that can produce high melatonin.

[0077] 2. Strain culture

[0078] Solid culture: Pick a small amount of bacteria and streak them on the corresponding solid plate, then invert and culture in a 30℃ constant temperature incubator for 1-2 days.

[0079] Liquid culture: Pick a small amount of bacteria and inoculate it into the corresponding liquid culture medium, and culture it in a constant temperature shaker at 30℃ and 220rpm for 15-20h.

[0080] 3. Yeast LiAc transformation

[0081] The specific method is as follows: a. Cultivate the yeast in YPDG liquid medium (containing 2% peptone, 1% yeast extract, 2% glucose and 2% glycerol) for 15-20 hours; b. Pipette 1mL of bacterial solution into a 1.5mL sterilized EP tube, centrifuge at 8000rpm for 45s, and discard the supernatant; resuspend the bacteria with 1mL ddH2O, centrifuge at 8000rpm for 45s, discard the supernatant, resuspend the bacteria with 1mL 0.1M lithium acetate buffer, and incubate in a 30℃ incubator for 1h. c. Centrifuge the above system at 8000rpm for 45s and discard the supernatant. Resuspend the cells with 70μL 0.1M lithium acetate buffer to obtain yeast competent cells; d. Add 240μL PEG4000 and 36μL lithium acetate buffer to the above system; e. Add 5μL salmon sperm DNA, 0.5-1μg transformation circular plasmid or linearized DNA fragment to the competent cells in sequence, and incubate at 30℃ for 25min. Heat shock at 42℃ for 30min; f. Centrifuge the above system at 8000rpm for 45s, remove part of the supernatant and resuspend the cells, add 600uLYPD liquid culture medium, incubate at 30℃ for 2-3h, spread on the solid plate with the corresponding screening marker, and invert and culture at 30℃ for 3-4 days; obtain the transformant of strain MT12.

[0082] 4. Yeast transformant colony PCR verification

[0083] Pipette 20 μL DNA lysis solution (MightyPrep reagent for DNA, TaKaRa) and dispense it into a PCR tube. Pick an appropriate amount of single colonies and mix them by pipetting in a PCR tube, then place them in a PCR instrument at 95°C for 10 minutes to lyse the bacteria. Take the supernatant as a template for colony PCR, select appropriate verification primers to prepare a PCR system, and verify the target gene.

[0084] Example 2

[0085] Application of the yeast engineering bacteria MT12 constructed by the present invention in biosynthesis of melatonin

[0086] 1. Effects of methyltransferases from five different animal and plant sources on melatonin synthesis by yeast engineered bacteria

[0087] 1. Construction of engineered strains

[0088] (1) According to the method described in Example 1, pJFE3-AtCOMT, pJFE3-MdASMT9, pJFE3-MeCOMT, pJFE3-OsCOMT, and pJFE3-HsCOMT plasmids were constructed respectively, and the cerevisiae strain CEN.PK 2-1c (genotype MATa; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2) was used as the starting strain. The above plasmids were transferred into the above starting strain by chemical transformation, and the corresponding engineered strains MT01-05 were obtained respectively. At the same time, the starting strain was used as the control strain.

[0089] (2) Shake flask fermentation

[0090] The transformants of strain MT01-05 (the transformants were prepared according to the method described in Example 1) were inoculated into 30 mL small white bottles containing 5 mL of SD+Ura liquid medium (8 g / L SD-Ura, 2% glucose) and cultured in a constant temperature shaker at 30°C and 220 rpm for 20-24 hours for activation. 1 mL of the activated culture solution was transferred to a 50 mL triangular flask containing 20 mL of SD-Ura fermentation medium and cultured at 30°C and 200 rpm for 72 hours. Samples were taken to detect the accumulation of melatonin in each group of fermentation products.

[0091] 2. Effects of methyltransferases from five different animal and plant sources on melatonin synthesis by yeast engineered bacteria

[0092] The results of melatonin accumulation in the fermentation product of strain MT01-05 are as follows Figure 1 As shown in A, compared with the control strain, after overexpressing 5 different methyltransferases in the starting strain, the accumulation of melatonin increased significantly, with significant differences (P<0.05); among them, the accumulation of melatonin synthesized by MdASMT9 from M. domestica in the engineered strain MT02 was significantly higher than that of the engineered strains MT01 and MT03-05. Under the condition of adding 100 mg / L of N-acetyl serotonin, the accumulation of melatonin synthesized by the engineered strain MT02 reached 21.7 mg / L ( Figure 1 B) The above results show that among the five methyltransferases from different animal and plant sources, N-acetyl serotonin methyltransferase ASMT from apple is the best choice.

[0093] 2. Construction of yeast engineering bacteria for the basic pathway of melatonin synthesis

[0094] Based on the screening of the best N-acetylserotonin methyltransferase ASMT, we constructed an engineered strain MT08 that simultaneously overexpresses arylalkylamine N-acetyltransferase, 5-hydroxytryptophan decarboxylase, tryptophan hydroxylase, and N-acetylserotonin methyltransferase. The specific method is: referring to the method described in Example 1, the pJ-MdASMT9-GgNNANT-HsDDC-GgTPH plasmid was constructed, and the cerevisiae strain was used as the starting strain. The above plasmid was transferred into the above starting strain by chemical transformation to obtain the engineered strain MT08.

[0095] The engineered strain MT08 transformant was subjected to shake flask fermentation test, and samples were taken for testing after 72 hours. The results of melatonin accumulation in the fermentation product of strain MT08 were as follows: Figure 2 As shown, after strain MT08 overexpressed arylalkylamine N-acetyltransferase, 5-hydroxytryptophan decarboxylase, tryptophan hydroxylase, and N-acetyl serotonin methyltransferase, the accumulation of melatonin reached 0.105 mg / L. After adding 1 g / L of the precursor tryptophan, the accumulation of melatonin reached 4.68 mg / L. The above results show that the yeast engineering bacteria for the basic pathway of synthesizing melatonin have been successfully constructed.

[0096] 3. Application of Yeast Engineering Bacteria MT12 in Biosynthesis of Melatonin

[0097] On the basis of determining the basic pathway for synthesizing melatonin, in order to enhance the synthesis of melatonin by yeast engineering bacteria, we constructed and optimized the synthesis and recycling pathway of tetrahydrobiopterin in the MT08 strain. Based on the M08 strain, we overexpressed 6-pyruvoyltetrahydrobiopterin synthase, septopterin reductase, 4a-hydroxytetrahydrobiopterin dehydratase, and dihydropterin reductase from laying hens to obtain the MT12 strain prepared in Example 1.

[0098] The engineered strain MT12 was tested by shake flask fermentation. Samples were taken for testing after 72 hours. The results were as follows: Figure 3 As shown, the melatonin accumulation of strain MT12 can reach 0.533 mg / L, which is about 5 times higher than that of strain MT08. After adding 1 g / L of the precursor tryptophan, the accumulation of melatonin in strain MT12 reached 14.32 mg / L. The above results show that the present invention constructs strain MT12 that can overexpress cofactors 6-pyruvoyltetrahydrobiopterin synthase, septopterin reductase, 4a-hydroxytetrahydrobiopterin dehydratase, and dihydropterin reductase on the basic pathway of synthesizing melatonin, which can significantly improve the ability and yield of yeast engineering bacteria to synthesize melatonin.

[0099] 4. 5L fermentation tank experiment of yeast engineering strain MT12

[0100] 1. Experimental methods

[0101] (1) Strain activation

[0102] The glycerol tube bacteria stored at -80°C were streaked on SD-Ura-His plates and inverted in a 30°C constant temperature incubator for 24 h.

[0103] (2) Primary seed culture

[0104] The mixed bacteria were picked from the plate and inoculated into a 50 mL Erlenmeyer flask containing 10 mL of SD-Ura-His liquid culture medium (2% peptone, 1% yeast powder, 2% glucose, 2% glycerol), and cultured in a constant temperature shaker at 30°C and 220 rpm for 20-24 h for activation.

[0105] (3) Secondary seed culture

[0106] The primary seeds were inoculated into a 2L shake flask containing 400mL SD-Ura-His medium at an inoculum size of 2%-4%, and cultured in a constant temperature shaker at 30°C and 220rpm for 20-24h.

[0107] (4) Fermentation in 5L fermenter

[0108] SD-Ura-His medium (2% tryptone, 1% yeast extract, 8% glucose) was used as the fermentation medium, with an initial volume of 9 L, a set temperature of 30°C, a ventilation volume of 0.5 vvm, a rotation speed of 400 rpm, and ammonia water was used to adjust the pH of the fermentation liquid to maintain at about 6.0. The secondary seeds were flame-inoculated in a 5L fermentation tank at an inoculation rate of 10%, and 4 g / L tryptophan and 1 g / L methionine were added in batches. Sampling tests were performed every 12 hours, and when the glucose concentration remained at 20 g / L, sugar addition was started.

[0109] 2. Detection method

[0110] (1) HPLC detection of compounds

[0111] Take 1 mL of sample, add 0.2 g of quartz sand and 1 mL of ethyl acetate, use a cell disruptor to disrupt for 10 min, centrifuge and absorb the supernatant, blow nitrogen at 80°C until dry, add 1 mL of methanol to dissolve, and filter into a liquid sample bottle using a 0.22 μm filter membrane. The concentrations of 5-hydroxytryptophan, serotonin, and N-acetyl serotonin were detected by WondaSil C18 column (GL Sciences) (4.6x250 mm) under HPLC UV-visible detector, with methanol and water as mobile phases, equal elution concentration, and analysis at a flow rate of 1 mL / min, column temperature set to 35°C, UV-visible detector wavelength 240 nm.

[0112] (2) Cell biomass detection

[0113] The fermentation broth was appropriately diluted according to the cell growth situation, and the value of 600nm wavelength was measured by UV spectrophotometer to obtain the OD value of cell growth. 600 .

[0114] 3. Results

[0115] The fermentation results are as follows Figure 4 As shown. The bacterial growth reached a stable stage after 32 hours, and OD 600 Maintained at around 30, melatonin continued to accumulate during the fermentation process. When the remaining glucose was 20g / L at 32h, sugar supplementation began. Finally, after 72h of fermentation, the accumulated melatonin reached 91.82mg / L.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A yeast engineering bacterium for synthesizing melatonin, characterized in that: The yeast engineering bacteria is constructed by a starting strain and genes of enzymes related to melatonin biosynthesis, wherein the starting strain is Saccharomyces cerevisiae; the enzymes related to melatonin biosynthesis include at least one of 5-hydroxy-L-tryptophan decarboxylase (DDC), arylalkylamine N-acetyltransferase (AANAT), N-acetylserotonin methyltransferase (AMST) / hydroxyindole O-methyltransferase (HIOMT) and tryptophan 5-hydroxylase (TPH).

2. The yeast engineered bacteria for synthesizing melatonin according to claim 1, characterized in that: The melatonin biosynthesis-related enzymes also include at least one of 6-pyruvoyl-tetrahydropterin synthase (6-pyruvoyl-tetrahydropterinsynthase, GgPTS), sepiapterin reductase (sepiapterin reductase, GgSPR), 4a-hydroxytetrahydrobiopterin dehydratase (4a-hydroxytetrahydrobiopterin dehydratase, GgPCBD1), and quinoid dihydropteridine reductase (quinoid dihydropteridine reductase, GgQDPR).

3. The yeast engineered bacteria for synthesizing melatonin according to claim 1 or claim 2, characterized in that: Among the enzymes related to melatonin biosynthesis, the N-acetylserotonin methyltransferase (AMST) / hydroxyindole O-methyltransferase (HIOMT) is derived from, including but not limited to, Arabidopsis thaliana, Malusdomestica, Oryza sativaL, Manihot esculenta and Homo sapiens; the arylalkylamine N-acetyltransferase is derived from Gallus gallus; the 5-hydroxytryptophan decarboxylase is derived from Homo sapiens; the tryptophan hydroxylase is derived from Gallus gallus; the 6-pyruvoyltetrahydrobiopterin synthase is derived from Gallus gallus; the septopterin reductase is derived from Gallus gallus; the dihydropteridine reductase is derived from Gallus gallus; and the 4a-hydroxytetrahydrobiopterin dehydratase is derived from Gallus gallus.

4. The method for constructing a yeast engineering bacterium for synthesizing melatonin according to claim 1, characterized in that: The construction method comprises the following steps: introducing the coding gene of the enzyme related to melatonin biosynthesis into the saccharomyces cerevisiae strain and over-expressing the gene.

5. The method for constructing a yeast engineering bacterium for synthesizing melatonin according to claim 4, characterized in that: Among the genes encoding enzymes related to melatonin biosynthesis, the N-acetylserotonin methyltransferase / hydroxyindole O-methyltransferase is shown in SEQ ID NO.1-5; the nucleotide sequence of the arylalkylamine N-acetyltransferase encoding gene is shown in SEQ ID NO.6; the nucleotide sequence of the 5-hydroxytryptophan decarboxylase encoding gene is shown in SEQ ID NO.7; the nucleotide sequence of the tryptophan hydroxylase encoding gene is shown in SEQ ID NO.8; the nucleotide sequence of the 6-pyruvoyltetrahydrobiopterin synthase encoding gene is shown in SEQ ID NO.9; the nucleotide sequence of the septopterin reductase encoding gene is shown in SEQ ID NO.10; the nucleotide sequence of the dihydropteridine reductase encoding gene is shown in SEQ ID NO.11; the nucleotide sequence of the 4a-hydroxytetrahydrobiopterin dehydratase encoding gene is shown in SEQ ID NO.

12.

6. The use of the yeast engineering bacteria in melatonin biosynthesis according to claim 1, characterized in that: The yeast engineered bacteria strain is fermented and cultured, and melatonin is separated and obtained.

7. Use of the yeast engineering bacteria according to claim 1 in preparing products rich in melatonin, wherein the products include foods, feed additives, health products, and medicines.

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