Genetically engineered bacterium, construction method thereof and application of genetically engineered bacterium in production of 5-hydroxytryptophan

Abstract

According to the invention, by optimizing the expression of related genes in the production path of recombinant microorganism 5-hydroxytryptophan and screening and strengthening the gene expression of 5-hydroxytryptophan to extracellular transport protein, the toxicity of product accumulation on thallus growth is greatly reduced. According to the present invention, the efficient synthesis of 5-hydroxytryptophan using glucose or glycerol as the carbon source is achieved, the 5-hydroxytryptophan is co-produced under the action of tryptophan hydroxylase by adding a part of tryptophan in the fermentation system as the substrate, and the conversion from the low-value carbon source to the high-added-value product is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a genetic engineering bacterium and an application thereof in producing 5-hydroxytryptophan. Background Art

[0002] 5-Hydroxytryptophan (5-HTP) is a tryptophan derivative, produced by tryptophan hydroxylase, and is also a precursor for the biosynthesis of serotonin and melatonin. 5-HTP is sold over-the-counter in the United States, the United Kingdom, and Canada as a dietary supplement for patients with depression, anorexia, and insomnia; it is also sold in many European countries as a drug for the treatment of major depressive disorder. Several placebo double-blind clinical trials have proven that 5-HTP is effective in treating depression, and the growing market demand has made 5-HTP one of the most commercially valuable small-breed amino acids.

[0003] At present, 5-HTP is mainly extracted from Ghana seeds, which requires crushing, filtration, centrifugation, decolorization, concentration, crystallization and other processes. The process is complicated and there is an unstable source of raw materials. Now there are chemical synthesis and microbial fermentation methods to produce 5-hydroxytryptophan, which have the advantages of low cost, simple process, and easy industrial production, becoming the first choice to replace plant extraction methods. However, most of the current fermentation production methods use tryptophan as a substrate to obtain the target product through biocatalysis. The disadvantage of this method is that it is necessary to first obtain cells with catalytic ability through fermentation, and then collect the cells for catalytic reaction. The operation is complicated and 5-hydroxytryptophan is all derived from tryptophan, which is costly. Although there are relevant literature reports on the synthesis of 5-hydroxytryptophan using glucose or glycerol as substrates, its maximum yield is still less than 10 g / L (Metabolicengineering, 2018, 48: 279-287; Microbial Cell Factories, 2022, 21(1): 198), which is far from meeting the needs of industrialization. There are many factors that limit the accumulation of 5-hydroxytryptophan during the fermentation process, such as the soluble expression and activity of tryptophan hydroxylase, the recycling and regeneration of cofactors, and the inability of intracellular 5-hydroxytryptophan to be transported out of the cell across the membrane in time, resulting in excessive intracellular concentration and toxic or inhibitory effects.

[0004] The discovery and engineering of transporters play an important role in the construction of cell factories. Reducing the intracellular concentration of the product can reduce product inhibition or toxicity. At the same time, reducing the intracellular product concentration is particularly beneficial for obtaining robust strains at high titers. However, it is challenging to identify transporters of exogenous chemicals in host strains. As the fermentation time accumulates, the accumulation of 5-hydroxytryptophan becomes slower and slower, which is speculated to be related to the intracellular inhibition of the product. However, there is no clear literature report indicating the transmembrane protein or regulatory transcription factor responsible for the transmembrane transport of 5-hydroxytryptophan. Therefore, it is speculated that the search for such a transmembrane transporter or transcription factor may have a certain promoting effect on the production of 5-hydroxytryptophan. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to solve the above technical problems, to design and construct a recombinant microorganism that can produce high 5-hydroxytryptophan, and to use the recombinant microorganism to synthesize 5-hydroxytryptophan using glycerol or glucose as a carbon source.

[0006] Technical solution: The genetically engineered strain of the present invention comprises genes for tryptophan hydroxylase, pterin-4a-methanol ammonia dehydratase and dihydropterin reductase synthesized by tetrahydrobiopterin, and genes for the regeneration cycle of tetrahydrobiopterin, including GTP cyclohydrolase I, guanosine reductase and 6-pyruvate tetrahydrobiopterin synthase.

[0007] Further, the GTP cyclohydrolase I is derived from Bacillus subtilis mtrA The 6-pyruvate tetrahydropterin synthase is encoded by the human PTPS gene, the guanosine reductase is encoded by the human SPR gene, the pterin-4a-methanol ammonia dehydratase is encoded by the human PCD gene, and the dihydropterin reductase is encoded by the human DHPR gene.

[0008] Furthermore, the nucleotide sequence of the tryptophan hydroxylase is shown in SEQ ID NO.1; the nucleotide sequence of the pterin-4a-methanol ammonia dehydratase is shown in SEQ ID NO.2, and the nucleotide sequence of the dihydropterin reductase is shown in SEQ ID NO.3.

[0009] Furthermore, the nucleotide sequences of the GTP cyclohydrolase I, 6-pyruvate tetrahydrobiopterin synthase and guanosine reductase are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0010] The present invention also provides a method for constructing a genetically engineered strain, comprising the following steps: 1) Construction of a genetically engineered strain for tryptophan synthesis; 2) Construction of 5-hydroxytryptophan synthesis strain: construct pTrc99A-TPD and pCS-MPS plasmids, and transform them into the strain prepared in step 1).

[0011] Furthermore, the transcription factor or transporter protein encoding genes arcA, arcB, Garp, aroP, and tnaB from Escherichia coli are expressed through a pCS-MPS plasmid to obtain an optimized genetically engineered strain.

[0012] Furthermore, it also includes expressing the related tyrosinase gene tyrA、tyrB and pheA The knockout step is performed on the obtained genetically engineered strain.

[0013] The present invention also provides the use of genetically engineered strains in the fermentation production of 5-hydroxytryptophan, using glucose or glycerol as a carbon source, adding part of tryptophan as a raw material, and the genetically engineered strains of the present invention are used as fermentation bacteria for fermentation production.

[0014] Furthermore, the fermentation conditions were as follows: the dissolved oxygen concentration was maintained at 20%-40%, the pH was controlled at 6.8-7.2, the aeration ratio was 1vvm, IPTG was used as an inducer, and the inducer concentration was 0.1-0.5 mM.

[0015] Beneficial effects: The genetically engineered strain of the present invention overexpresses tryptophan hydroxylase and PCD and DHPR genes synthesized by tetrahydrobiopterin on the genome or plasmid; in order to improve the synthesis efficiency of tetrahydrobiopterin, the regeneration cycle genes mtrA, spr, ptps of tetrahydrobiopterin are overexpressed; in order to reduce the degradation of the target product 5-hydroxytryptophan and the precursor tryptophan, the CRISPR / Cas9 system is used to knock out the 5-hydroxytryptophan on the genome. tnaA Gene; In order to reduce the accumulation of intracellular 5-hydroxytryptophan and enhance the efflux capacity of 5-hydroxytryptophan in the engineered strain, a gene with 5-hydroxytryptophan transport function is expressed through a plasmid or genome. The present invention optimizes the expression of related genes in the 5-hydroxytryptophan production pathway of recombinant microorganisms and screens and strengthens the gene expression of 5-hydroxytryptophan transport proteins to the extracellular space, thereby greatly reducing the toxicity of product accumulation to bacterial growth. The efficient synthesis of 5-hydroxytryptophan with glucose or glycerol as the carbon source is achieved, and at the same time, part of tryptophan is added as a substrate in the fermentation system, and 5-hydroxytryptophan is co-produced under the action of tryptophan hydroxylase, thereby realizing the transformation of low-value carbon sources into high-value-added products.

[0016] By utilizing the genetically engineered strain and optimized fermentation conditions provided by the present invention, efficient synthesis of 5-hydroxytryptophan is achieved mainly through modular optimization of gene expression, screening of transport proteins, increasing the concentration of target products, and reducing the toxicity of products to strains. The concentration of 5-hydroxytryptophan in shake flask fermentation can reach 5.8 g / L, and the production of 5-hydroxytryptophan (5-HTP) after 65 h of fermentation with glucose as a carbon source reaches 20.9 g / L, and the production of 5-hydroxytryptophan (5-HTP) after 65 h of fermentation with glycerol as a carbon source reaches 22.3 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a fermentation production process diagram of Example 6 of the present invention.

[0018] Figure 2 It is a fermentation production process diagram of Example 7 of the present invention. DETAILED DESCRIPTION

[0019] The following is a specific implementation method in the form of embodiments to further illustrate the contents of the present invention in detail, but this should not be understood as all the contents of the present invention. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0020] Example 1: Construction of a chassis strain for enhancing tryptophan synthesis In this example, the Escherichia coli strain was selected E . coli W3110 is a production strain that uses CRISPR gene editing technology to modify the tnaA Gene knockout blocks the degradation pathway of tryptophan and 5-hydroxytryptophan in E. coli. trpL Knock out and reduce trpE Feedback inhibition strengthens genes trpE fbrS40F The expression (Δ trpL ::pTrc- trpE fbrS40F ); knockout tyrR While strengthening aroG fbr Gene expression (Δ tyrR ::pTrc- aroG fbrS211F ), to improve the accumulation of tryptophan, and the tryptophan production in shake flasks reached 2.5 g / L.

[0021] The knockout construction adopts the CRISPR gene editing method. tnaA The knockout is described in detail.

[0022] 1. First, prepare the competent state of Escherichia coli W3110 strain. The specific operations are as follows: (1) Inoculate a single clone of E. coli W3110 into a test tube containing 5 mL of LB medium and culture overnight at 37°C, 200 rpm / min.

[0023] (2) Inoculate 50 mL LB medium at a 1% inoculum volume and culture at 37°C until OD 600 About 0.6 (about 2 h).

[0024] (3) Transfer the bacterial solution to a 50 mL pre-cooled centrifuge tube, place on ice for 20 min, and centrifuge at 4°C, 4000 rpm / min for 5 min.

[0025] (4) Discard the supernatant and add 15 mL of pre-cooled 10% glycerol solution to suspend the bacteria. Centrifuge at 4°C, 4000 rpm / min for 5 min.

[0026] (5) Repeat (4) twice.

[0027] (6) Discard the supernatant and add 500 μl of pre-cooled 10% glycerol solution to gently suspend the bacteria. Then, aliquot 100 μl / tube into sterilized 1.5 ml centrifuge tubes and store in a -80°C refrigerator until use.

[0028] 2. Prepare the recombinant E. coli W3110 strain containing pCas9 plasmid by electroporation: (1) Add 2 μl of pCas9 plasmid to 100 μl of W3110 competent bacteria, mix gently, and incubate on ice for 20 min.

[0029] (2) Wipe the side of the pre-cooled 0.2 cm electroporation cuvette clean of moisture, add all the competent cells into the cuvette, and perform electroporation at 2.5 KV.

[0030] (3) After the electroporation, add 700 μl of LB medium without antibiotics and incubate at 30°C for 1 h to allow the cells to recover.

[0031] (4) Spread the bacteria evenly on an LB plate containing kanamycin resistance and culture in a 30°C incubator for 16 h.

[0032] (5) Pick a single colony for colony PCR and verify the correct positive clone for subsequent knockout construction.

[0033] 3. Construction for knockout tnaA pTarget- tnaA Plasmid and donor fragment, the specific operation is: Using the original pTarget plasmid as a template and pTarget-TnaA-F / pTarget-R as primers, PCR amplification was performed to replace the N20 sequence on the original pTarget plasmid with a targetable tnaA The N20 sequence of the gene was amplified and the fragment was recovered from the gel. The recovered fragment was demethylated with Dpn I restriction endonuclease to remove the original plasmid template, introduced into Top10 competent medium, placed on ice for 20 min, heat-shocked at 42°C for 90 s, placed on ice for 5 min, added with 700 μl of LB culture medium without resistance, incubated at 37°C for 45 min, and then coated on LB plates containing spectinomycin resistance.

[0034] The Donor segment contains tnaA The upstream and downstream homology arms of the gene are used tnaA Repair of genes cut by Cas9. The construction method is to use the W3110 genome sequence as a template, TnaA-up-F / TnaA-up-R as primers to amplify the upper homology arm TnaA-up; TnaA-dm-F / TnaA-dm-R as primers to amplify the lower homology arm TnaA-dm, and gel-recover the product. Using the two fragments of TnaA-up and TnaA-dm as templates, TnaA-up-F / TnaA-dm-R as primers to perform overlapping PCR, splice the upstream and downstream homology arms, and gel-recover the fragments, which are used as donors for TnaA gene knockout.

[0035] 4. Prepare competent E. coli W3110 containing pCas9 plasmid. The specific preparation method is as follows: (1) Inoculate a single clone of E. coli W3110 containing the pCas9 plasmid into an LB tube supplemented with kanamycin resistance and culture overnight at 30°C, 200 rpm / min.

[0036] (2) Inoculate 50 mL of LB medium at a 1% inoculum concentration and culture at 30°C until OD 600 When the OD was about 0.2, 600 μl of 1 M arabinose solution was added and the culture was continued until the OD 600 About 0.6.

[0037] (3) Transfer the bacterial solution to a 50 mL pre-cooled centrifuge tube, place on ice for 20 min, and centrifuge at 4°C, 4000 rpm / min for 5 min.

[0038] (4) Discard the supernatant and add 15 mL of pre-cooled 10% glycerol solution to suspend the bacteria. Centrifuge at 4°C, 4000 rpm / min for 5 min.

[0039] (5) Repeat (4) twice.

[0040] (6) Discard the supernatant and add 500 μl of pre-cooled 10% glycerol solution to gently suspend the bacteria. Then, dispense 100 μl / tube into sterilized 1.5 ml centrifuge tubes and store in a -80°C refrigerator until use.

[0041] 5. Add 2 μl of pTarget-TnaA plasmid and 400 ng of donor fragment to 100 μl of E. coli W3110 competent cells containing pCas9 plasmid, mix gently, and place on ice for 20 min. Perform electroporation at 2.5 KV, add 700 μl of LB culture medium without antibiotics, incubate at 30°C for 1 h, apply LB plates containing kanamycin and spectinomycin double resistance, and place in a 30°C incubator for 16 h.

[0042] Single clones on the plate were picked for colony PCR verification and then sent for sequencing. The positive clones with correct sequencing were inoculated into LB tubes containing 0.2 mM IPTG and kanamycin resistance and cultured at 30°C overnight to remove the pTarget plasmid; the single clones with the pTarget plasmid removed were inoculated into LB tubes without resistance and cultured at 42°C overnight to remove the pCas9 plasmid. Finally, the knockout of Cas9 in W3110 was obtained. tnaA coli recombinant strains of the gene.

[0043] pTrc-trpED fbr Integration in W3110, and ΔtyrR::pTrc- aroG fbr The knock-in method uses CRISPR gene editing technology. The specific process refers to the knock-out of tnaA. The primer sequences involved in the knock-in process are shown in Table 1. tnaA , overexpression tprED fbr and aroG fbr The recombinant Escherichia coli after gene editing was named SH01.

[0044] Table 1: Construction of tryptophan accumulation genetic engineering strains

[0045] Example 2: Expression optimization of key genes for the biotransformation of tryptophan into 5-hydroxytryptophan and its effects In order to balance the flux of different modules in the tryptophan to 5-hydroxytryptophan synthesis pathway, and then optimize the 5-hydroxytryptophan synthesis pathway and the module gene expression intensity, this part uses high-copy plasmid pTrc99a (pBR322-colEori), medium-copy plasmid pBbA1a (pBR322-p15A ori), and low-copy plasmid pCS (pBR322-psc101 ori); first, the two modules hTPH2-PCD-DHPR (TPD for short) and mtrA-PTP-SPR (MPS for short) are placed on high-, medium-, and low-copy vectors for expression, respectively. The molecular biology process includes: PCR polymerase chain reaction, enzyme digestion, ligation, electrochemical conversion, etc.

[0046] This example mainly describes the construction of pTrc99A-TPD and pCS-MPS. pTrc99A-TPD is a plasmid pTrc99A with three gene fragments of TPH2, PCD and DHPR inserted into it. pCS-MPS is a plasmid pCS with three gene fragments of mtrA, PTPS and SPR inserted into it. All genes on the plasmid are expressed by the pTac promoter.

[0047] The plasmid was constructed by one-step cloning. The construction of this plasmid was completed by one-step construction. pTrc99A-TPD was constructed by inserting three gene fragments of TPH2, PCD and DHPR into the pTrc99A plasmid using a one-step cloning kit. The construction method of pCS-MPS was the same as above. The kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The construction process was referred to the instructions of the one-step cloning kit.

[0048] First, the pTrc99A plasmid was Eco After single digestion with RI restriction endonuclease, the linearized plasmid was recovered. TPH2, PCD and DHPR fragments were PCR amplified from the synthesized TPH2, PCD and DHPR gene fragments using TPH2-F / TPH2-R, PCD-F / PCD-R and DHPR-F and DHPR-R as primers, respectively. The fragments were recovered by gel, and the use of enzyme digestion and gel recovery reagents was carried out according to the reagent instructions.

[0049] Secondly, the pCS plasmid was also Eco RI and XhoIAfter single restriction enzyme digestion, the linearized plasmid was recovered. The mtrA, PTPS and SPR fragments were PCR amplified from the synthesized TPH2, PCD, and DHPR gene fragments using mtrA-F / mtrA-R, PTPS-F / PTPS-R and SPR-F and SPR-R as primers, respectively. The fragments were recovered by gel, and the enzyme digestion and gel recovery reagents were used according to the reagent instructions.

[0050] After the construction of pTrc99A-TPD and pCS-MPS plasmids was completed, sequencing was performed to ensure the accuracy of the inserted sequence. The primers are shown in Table 2.

[0051] Table 2 shows the primer sequences involved in this example.

[0052] (2) The plasmid successfully constructed in the above steps was transformed into the SH01 strain to obtain the engineered strain as shown in Table 3. The obtained engineered strain was fermented by shake flask fermentation. The shake flask fermentation medium was as follows: yeast powder 6 g / L, potassium dihydrogen phosphate 5.5 g / L, citric acid monohydrate 0.5 g / L, magnesium sulfate heptahydrate 3 g / L, ammonium sulfate 5 g / L, glucose 15 g / L, pyridoxal phosphate 1 mg / L, vitamin B1 1 mg / L, calcium pantothenate 1 mg / L, 1 mg / L HBO3, 1 mg / L CuCl2, 1 mg / L CoCl2, 2 mg / L ZnCl2, 2 mg / L MnCl2, 5 mg / L FeSO4. The engineered bacterial single clone was inoculated into 5 mL LB liquid medium and cultured overnight (37 °C, 220 rpm) until the OD 600 When the OD value reaches 2-3, transfer to fermentation medium with an inoculation volume of 2%. In the early stage, culture at 37°C and 220 rpm. 600When the pH value reached 0.6-0.8, IPTG was added at a final concentration of 0.2 mM, and the mixture was placed under 30°C and 220 rpm conditions for fermentation and culture. The fermentation cycle was 60 h. After the fermentation, the contents of tryptophan and 5-hydroxytryptophan were determined by high performance liquid chromatography: 1 ml of the fermentation broth was centrifuged (12,000 rpm, 5 min), the supernatant was diluted 10-50 times according to the concentration of the substance to be determined, and then filtered through a 0.22 μm filter membrane. The filtrate was detected by an Agilent -HPLC 1260 liquid phase system. The chromatographic column was Halo RP-Amide C18 (4.6*150 mm, 2.7 μm), the detection wavelength was 280 nm; the column temperature was set to 35°C; the flow rate was 1.0 mL / min; the injection volume was set to 5.0 μL, and the mobile phase was methanol: 0.1% formic acid water (v / v) = 5:95. 5-hydroxytryptophan was produced, and the yield results are shown in Table 3.

[0053] Table 3: Effect of module-optimized balanced pathway flux on 5-hydroxytryptophan production

[0054] The results of shake flask fermentation showed that in the synthesis pathway of 5-hydroxytryptophan using tryptophan as a substrate, the expression levels of different genes had a great influence on the yield of 5-hydroxytryptophan. Among them, TPD was expressed on medium and high copy vectors, and the yield was significantly improved. The final experimental results showed that the expression of TPD in high copy and MPS in low copy combination had the best effect, and the yield of 5-hydroxytryptophan reached 2.3 g / L in 60 h of fermentation.

[0055] Example 3: Screening of transporters and transcription factors to improve the accumulation of target products Strengthen transport proteins or transcription factors, increase the extracellular transport of 5-HTP, and improve the accumulation of target products; based on the high-yield 5-hydroxytryptophan strain SH02, increase the extracellular transport of 5-hydroxytryptophan to improve the accumulation of products; strengthen the expression of transport proteins, optimize the integration of plasmids and genomes, and test the fermentation effect; preferably express strong promoters on plasmids arcA , arcB , Garp , aroP and tnaB Gene; The specific operation of plasmid construction is as follows: pCS-MPS plasmid is digested with XhoI restriction endonuclease, and the linearized plasmid is recovered. arcA, arcB, Garp, aroP, and TnaB fragments are amplified from the genome of Escherichia coli MG655 using arcA-F / arcA-R, arcB-F / arcB-R, Garp-F / Garp-R, aroP-F / aroP-R, and TnaB-F / TnaB-R as primers, respectively. The fragments are recovered by gel, and the use of enzyme digestion and gel recovery reagents can be operated according to the reagent instructions.

[0056] After the construction of pCS-MPSA, pCS-MPSB, pCS-MPSG, pCS-MPSP and pCS-MPST plasmids was completed, sequencing was performed to ensure the accuracy of the inserted sequence. The primers are shown in Table 4.

[0057] Table 4: Primer sequences involved in this Example 3

[0058] The plasmid successfully constructed in the above steps and the plasmid pTrc99A-TPD were transformed into the SH01 strain to obtain the engineered strain as shown in Table 5. The obtained engineered strain was used to produce 5-hydroxytryptophan by shake flask fermentation. The fermentation operation was the same as in Example 2, and the yield results are shown in Table 5.

[0059] Table 5: Fermentation results of engineered bacteria with enhanced transporter protein expression:

[0060] The results of shake flask fermentation are shown in Table 5. The enhanced expression of GarP can effectively promote the biosynthesis of 5-hydroxytryptophan, proving that the use of transporters can promote the transport of products out of the cell, thereby reducing cytotoxicity is an effective strategy. Among them, strain SH09 has the best effect, making the 5-hydroxytryptophan production reach 3.08 g / L, and the production of 5-hydroxytryptophan increased by 40%.

[0061] Example 4 The yield and extraction rate of 5-HTP were improved by metabolic engineering of the strain.

[0062] Tyrosine and tryptophan are both aromatic amino acids. At the same time, tyrosine can be converted into L-dopa and melanin, leading to the accumulation of pigment substances. In order to further increase the proportion of tryptophan produced by the engineered strain and facilitate the subsequent extraction of the target product 5-hydroxytryptophan, the relevant tyrosinase gene was expressed. tyrA、tyrB and pheA Knockouts are performed to allow more carbon to flow into the synthetic pathway of the target product.

[0063] Specific implementation method: Knockout construction uses the Crispr-Cas9 gene editing method, for specific examples, refer to tnaA The knockout process, primers are shown in Table 6.

[0064] Table 6: Primer sequences for modification of tyrosine synthesis pathway

[0065] The engineered strains successfully constructed in the above steps were transformed with plasmids pTrc99A-TPD and pCS-MPSG to obtain the engineered strains shown in Table 7. The obtained engineered strains were fermented in shake flasks to produce 5-hydroxytryptophan. The fermentation operation was the same as in Example 2. The yield of 5-hydroxytryptophan is shown in Table 7. The results showed that the knockout tyrA The genetically engineered strain SH12 had the highest production of 5-hydroxytryptophan.

[0066] Table 7: Details of engineered strains with reduced tyrosine accumulation

[0067] Example 5: Fermentation Condition Optimization - Effect of Inducer IPTG Concentration The engineered strain SH12 was optimized for shake flask fermentation, and the IPTG addition concentration was optimized. 0.1 mM, 0.15 mM, 0.2 mM, and 0.5 mM IPTG concentrations were selected for shake flask fermentation tests. The results showed that 0.1 mM had the best effect, and the 5-hydroxytryptophan production was increased by about 20% compared with the previous 0.2 mM condition. The final shake flask test results are shown in Table 8. The highest 5-hydroxytryptophan concentration reached 5.8 g / L; the results showed that low concentration IPTG was more conducive to the growth of Escherichia coli cells and the expression of hydroxylase.

[0068] Table 8: Optimization of IPTG concentration

[0069] Example 6: Fermentation production of 5-hydroxytryptophan 5-HTP was produced by fed-batch fermentation in a 5-L fermenter using glucose as the carbon source.

[0070] The fermentation medium composition includes: 1-20g / L yeast powder, 1-15 g / L KH2PO4, 1-5 g / L MgSO4, 1-10g / L(NH4)2SO4, 0.01-1 mg / L HBO3, 0.1-1 mg / L CuCl2, 0.1-1 mg / L CoCl2, 0.5-2 mg / LZnCl2, 1-3 mg / L MnCl2, 1-5 mg / L FeSO2, 0.5-5 mg / L pyridoxal phosphate, 0.2-1 mg / L vitamin B1, 0.2-1 mg / L calcium pantothenate, 5-30 g / L glucose or 5-50 g / L glycerol.

[0071] Fermentation conditions: The engineered strain SH12 was inoculated into LB liquid medium containing ampicillin and kanamycin sulfate and cultured overnight at 37°C. A 1% inoculum was transferred to 200 ml fresh LB medium as a secondary seed culture. When the OD 600 When the OD reaches about 4, the secondary seed solution is inoculated into the fermenter at a 10% inoculation rate for expansion culture. The fermenter medium formula is the same as the shake flask fermentation medium formula, and the initial culture temperature is 37°C. During the fermentation process, the dissolved oxygen concentration is maintained at 20%-40%, the pH is controlled at 6.8-7.2, ammonia water is used as the pH regulator, and the ventilation ratio is 1 vvm. When the OD 600 When the temperature reached 25, the temperature was slowly lowered to 30℃, and 5 g / L tryptophan was added at the same time. The IPTG induction concentration was 0.1 mM. Tryptophan was fed as a substrate during the fermentation process to maintain the tryptophan (TRP) concentration at 2-5 g / L. After the initial glucose was consumed and the dissolved oxygen rebounded, carbon source feeding (600 g / L glucose) was started. During the fermentation process, the glucose concentration in the fermentation system was maintained at a low level (1-2 g / L) until the end of the fermentation.

[0072] Take samples regularly during the fermentation process to measure the bacterial OD 600 , 1 ml of the fermentation broth was centrifuged and the supernatant was diluted to an appropriate concentration. The supernatant was filtered through a 0.22 μm membrane. During the fermentation process, HPLC was used to detect the content of tryptophan, 5-hydroxytryptophan and the concentration of glucose.

[0073] The fermentation results are as follows Figure 1 As shown in the figure, with the extension of fermentation time, OD increased continuously and finally reached 110. By adding some tryptophan exogenously, the tryptophan concentration was maintained at about 4 g / L during the fermentation process. 5-HTP accumulated continuously during the whole fermentation process. Finally, after 65 h of fermentation, the 5-HTP production reached 20.9 g / L.

[0074] Example 7: Fermentation production of 5-hydroxytryptophan Glycerol (Gly) was used as the carbon source, and other conditions were consistent with those in Example 6. The fermentation results were as follows: Figure 2 As shown in the figure, with the extension of fermentation time, OD increased continuously and finally reached 125. By adding some tryptophan exogenously, the tryptophan concentration was maintained at about 4 g / L during the fermentation process, and 5-HTP accumulated continuously. Finally, after 65 h of fermentation, the 5-HTP production reached 22.3 g / L.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A genetically engineered strain, characterized in that: It contains genes for tryptophan hydroxylase, pteridine-4a-carbinol ammonia dehydratase and dihydropterin reductase synthesized by tetrahydrobiopterin, as well as the regeneration cycle genes GTP cyclohydrolase I, guanosine reductase and 6-pyruvate tetrahydrobiopterin synthase of tetrahydrobiopterin.

2. The genetically engineered strain according to claim 1, characterized in that The GTP cyclohydrolase I is encoded by the mtrA gene from Bacillus subtilis, the 6-pyruvate tetrahydrobiopterin synthase is encoded by the human PTPS gene, the guanosine reductase is encoded by the human SPR gene, the pterin-4a-methanol ammonia dehydratase is encoded by the human PCD gene, and the dihydropterin reductase is encoded by the human DHPR gene.

3. The genetically engineered strain according to claim 1, characterized in that The nucleotide sequence of the tryptophan hydroxylase is shown in SEQ ID NO.1; the nucleotide sequence of the pterin-4a-methanol ammonia dehydratase is shown in SEQ ID NO.2, and the nucleotide sequence of the dihydropterin reductase is shown in SEQ ID NO.

3.

4. The genetically engineered strain according to claim 1, characterized in that The nucleotide sequences of the GTP cyclohydrolase I, 6-pyruvate tetrahydrobiopterin synthase and guanosine reductase are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

5. The method for constructing a genetically engineered strain according to claim 1, characterized in that: The following steps are involved: 1) Construction of a genetically engineered strain for tryptophan synthesis; 2) Construction of 5-hydroxytryptophan synthesis strain: construct pTrc99A-TPD and pCS-MPS plasmids, and transform them into the strain prepared in step 1).

6. The method for constructing a genetically engineered strain according to claim 5, characterized in that: The transcription factor or transport protein encoding genes arcA, arcB, Garp, aroP and TnaB derived from Escherichia coli are expressed through a pCS-MPS plasmid to obtain an optimized genetic engineering strain.

7. The method for constructing a genetically engineered strain according to claim 5 or 6, characterized in that: Tyrosinase gene expression tyrA、tyrB and pheA The knockout step is performed on the obtained genetically engineered strain.

8. Use of the genetically engineered strain according to any one of claims 1 to 7 in the fermentation production of 5-hydroxytryptophan.

9. The use according to claim 8, characterized in that: Glucose or glycerol is used as a carbon source, some tryptophan is added as a raw material, and the genetically engineered strain according to any one of claims 1 to 4 is used as a fermentation bacterium for fermentation production.

10. The use according to claim 9, characterized in that: The fermentation conditions were as follows: the dissolved oxygen concentration was maintained at 20%-40%, the pH was controlled at 6.8-7.2, the aeration ratio was 1 vvm, IPTG was used as an inducer, and the inducer concentration was 0.1-0.5 mM.

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