Genetically engineered bacterium and method for producing 5-hydroxytryptophan through whole-cell catalysis
Through genetic engineering, E. coli was constructed, and the engineering bacteria TPH-2 was used, and whole-cell catalytic technology was used to solve the problem that the yield and cost of the existing microbial fermentation method were difficult to take into account both in the production of 5-hydroxytryptophan, and achieved efficient and high-yield conversion of 5-hydroxytryptophan under mild conditions, which was suitable for industrial production.
Patent Information
- Application Number
- CN202510260503.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-05-30
AI Technical Summary
When the existing microbial fermentation method produces 5-hydroxytryptophan, it cannot take into account both the output and cost reduction, resulting in industrial production being unable to meet market demand.
Escherichia coli was genetically engineered, and the tryptophan gene tnaA was knocked out, and genes such as tryptophan hydroxylase, GTP cyclization hydrolase I, pterin-4α-methanolamine dehydrase, mepterin reductase, dihydropterin reductase and 6-acetone tetrahydropterin synthase were introduced to construct the engineered bacteria TPH-2, and L-tryptophan was converted to 5-hydroxytryptophan under mild conditions using whole-cell catalytic technology.
It has achieved efficient and high yield conversion of L-tryptophan into 5-hydroxytryptophan under mild conditions, with higher yields, a conversion rate of 55.7%, and a production intensity of 0.116g/L/h, which is suitable for industrial production.
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Abstract
Description
Technical Field:
[0001] The present invention belongs to the technical field of biochemistry engineering, and particularly relates to a genetically engineered bacterium and a method for producing 5-hydroxytryptophan by whole-cell catalysis. Background Art:
[0002] 5-Hydroxytryptophan is a biochemical substance with important physiological activities, and can be used for synthesizing neurotransmitter serotonin, hormone melatonin, etc., and has broad application prospects in the fields of medicine, food, feed, etc. At present, the production methods of 5-hydroxytryptophan mainly include chemical synthesis method and plant extraction method. The chemical synthesis method has harsh reaction conditions and high costs; the plant extraction method is limited by the raw material sources and has limited yields.
[0003] The microbial fermentation method provides a new way for the production of 5-hydroxytryptophan. Some studies have achieved the biosynthesis of 5-hydroxytryptophan by constructing recombinant microorganisms and expressing related enzyme genes. For example, Escherichia coli as a host bacterium, expressing tryptophan hydroxylase and the tetrahydrobiopterin synthesis and regeneration pathway, can catalyze the conversion of L-tryptophan into 5-hydroxytryptophan. At present, the literature reports that the highest yield of de novo synthesis of 5-hydroxytryptophan by Escherichia coli cell factory using glucose as a substrate is only 8.58 g / L, and its synthesis of BH by introducing a heterologous BH 4 synthesis pathway to synthesize BH 4; Despite the high yield, there are still the following defects: the conversion rate of 5-hydroxytryptophan to glucose is only 0.095 g / g, which is not suitable for industrial production. (Metabolic engineering of Escherichia coli for efficient production of L-5-hydroxytryptophan from glucose. Microbial Cell Factories, 2022, 21, 198.). In addition, using 2 g / L tryptophan as the substrate, 1.302 g / L of 5-hydroxytryptophan can be synthesized, and the conversion rate to tryptophan is 65.1%. However, this study requires the addition of a high concentration of whey powder, increasing the production cost (Efficient synthesis of 5-hydroxytryptophan in Escherichia coli by bifunctional utilization of whey powder as a substrate for cell growth and inducer production. Journal of Biotechnology, 2024, 393, 100 - 108.). It has the following defects: the fermentation period is 72 h, the production intensity is only 0.018 g / L / h, a high concentration of whey powder (80 g / L), and the high cost makes it impossible to be applied industrially.
[0004] Therefore, in the existing microbial fermentation method, it is impossible to balance the yield and cost reduction of 5-hydroxytryptophan, resulting in the inability of the industrial production of 5-hydroxytryptophan to meet the market demand.
[0005] The whole-cell catalysis technology is a technology that uses the enzyme system of microbial cells themselves to carry out catalytic reactions, and has the advantages of mild reaction conditions, high selectivity, environmental friendliness, etc. Applying the whole-cell catalysis technology to the production of 5-hydroxytryptophan is expected to improve production efficiency and reduce production costs, and has important application value. Summary of the Invention:
[0006] Object of the Invention:
[0007] The object of the present invention is to provide a new method that is efficient, high-yield, and can use L-tryptophan to convert and produce 5-hydroxytryptophan under mild conditions to meet the market demand for 5-hydroxytryptophan.
[0008] Technical Solution:
[0009] The first object of the present invention is to provide a genetically engineered bacterium, which is obtained by genetically engineering the starting strain Escherichia coli w3110; the genetic engineering is to not express the tryptophanase gene tnaA. The non-expression includes knockout or silencing.
[0010] In the present invention, the tryptophan hydroxylase gene Tph from Homo, the GTP cyclohydrolase I gene GchI from artificial synthesis, the pterin-4α-carbinolamine dehydratase gene Pcd from Homo, the sepiapterin reductase gene Spr from Homo, the dihydropteridine reductase gene Dhpr from Homo, and the 6-pyruvoyltetrahydropterin synthase gene Ptps from Homo are introduced into Escherichia coli. It can efficiently biotransform L-tryptophan to produce 5-hydroxytryptophan.
[0011] In one embodiment of the present invention, the nucleotide sequence of the tryptophan hydroxylase gene Tph is as shown in NO.1, the nucleotide sequence of the GTP cyclohydrolase I gene GchI is as shown in NO.2, the nucleotide sequence of the pterin-4α-carbinolamine dehydratase gene Pcd is as shown in NO.3, the nucleotide sequence of the sepiapterin reductase gene Spr is as shown in NO.4, the nucleotide sequence of the dihydropteridine reductase gene Dhpr is as shown in NO.5, and the nucleotide sequence of the 6-pyruvoyltetrahydropterin synthase gene Ptps is as shown in NO.6.
[0012] In one embodiment of the present invention, the tnaA gene (Sequence ID: CP062250.1) is knocked out using the Crispr-Cas9 system.
[0013] The present invention also provides a method for preparing the recombinant Escherichia coli, comprising the following steps:
[0014] (1) Connect the genes in the order of Spr, Dhpr, and Ptps to the plasmid pEM to construct the recombinant plasmid pEM-Spr-Dhpr-Ptps;
[0015] (2) Connect the genes in the order of Tph, GchI, and Pcd to the plasmid pET28a-T5 to construct the recombinant plasmid pET28a-T5-Tph-GchI-Pcd;
[0016] (3) Transform the recombinant plasmid pEM-Spr-Dhpr-Ptps constructed in step (1) and the recombinant plasmid pET28a-T5-Tph-GchI-Pcd constructed in step (2) into the competent cells of Escherichia coli with the tnaA gene knocked out to obtain the recombinant Escherichia coli.
[0017] The present invention also provides a method for preparing a cell catalyst for producing 5-hydroxytryptophan, comprising fermenting the recombinant Escherichia coli and collecting the cells.
[0018] In one embodiment of the present invention, the fermentation is carried out by culturing the recombinant Escherichia coli in LB medium at 37 °C with a rotation speed of 200 rpm for 8 - 10 h, then transferring it to TB medium and culturing at 37 °C with a rotation speed of 200 rpm until OD 600 = 0.6 - 0.8, adding IPTG, and simultaneously adding tryptophan for whole-cell catalysis.
[0019] The present invention also provides any one of the following applications of the above recombinant microorganism, its preparation method, the method for preparing a cell catalyst for producing 5-hydroxytryptophan, and the method for whole-cell catalytic production of 5-hydroxytryptophan:
[0020] (1) Application in the fermentation production of 5-hydroxytryptophan;
[0021] (2) Application in improving the ability of biological synthesis of 5-hydroxytryptophan.
[0022] Principle of the present invention
[0023] As Figure 1 shown, in the synthesis pathway of 5-hydroxytryptophan, tryptophan hydroxylase (TPH) uses tetrahydrobiopterin (BH 4 ) and Fe 2+ as cofactors to catalyze the synthesis of 5-hydroxytryptophan from tryptophan. Among them, using GTP as a substrate, it is catalyzed by GTP cyclohydrolase I (GCHI) to synthesize 7,8-dihydroneopterin triphosphate; then catalyzed by 6-pyruvoyltetrahydropterin synthase (PTPS) to synthesize 6-pyruvoyl-5,6,7,8-tetrahydropterin; finally, catalyzed by sepiapterin reductase (SPR) to synthesize BH 4 , constituting the BH 4 regeneration pathway. Pterin-4α-carbinolamine is catalyzed by pterin-4α-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) to synthesize BH 4 , constructing the BH 4 cycle module. In Escherichia coli cells, tryptophanase can degrade tryptophan into indole and pyruvate, which is a competing metabolic branch in the 5-hydroxytryptophan synthesis pathway. Therefore, in constructing an Escherichia coli cell factory for 5-hydroxytryptophan, the tnaA gene is knocked out using the Crispr-Cas9 system to block this competing metabolic branch.
[0024] Beneficial effects:
[0025] The present invention provides a method for producing 5-hydroxytryptophan by whole-cell catalysis, which catalyzes the synthesis of endogenous BH through a designed synthetic route 4 without the need to add expensive exogenous BH 4 ; all the enzymes involved in the present invention are codon-optimized (such as Spr, Dhpr, Ptps, Tph, GchI, Pcd genes), which are different from the enzyme sequences in the prior art, and the catalytic efficiency is greatly increased; the dual plasmids pEM-Spr-Dhpr-Ptps and pET28a-T5-TphI-GchI-Pcd are used, and the tnaA gene is knocked out simultaneously to block this competitive metabolic branch, and the engineering bacterium TPH-2 (E. coli w3110ΔtnaA pEM-Spr-Dhpr-Ptps and pET28a-T5-TphI-GchI-Pcd) is constructed. The design of this dual plasmid has not been reported in the literature, and it is simple and easy for industrial application. The present invention can use L-tryptophan to convert and produce 5-hydroxytryptophan under mild conditions through TPH-2. The reaction time is shorter than that of direct fermentation (48 h), and there are fewer intermediate operations and higher yields. Using L-tryptophan as a substrate, adding recombinant Escherichia coli, and using ordinary LB medium, whole-cell catalysis is carried out at 30 °C, consuming 10 g / L of L-tryptophan, and 5.57 g / L of 5-hydroxytryptophan can be produced without exogenous addition of NADPH (and without the need to add whey powder), the conversion rate reaches 55.7%, and the production intensity is 0.116 g / L / h, which is suitable for industrial production. Description of the Drawings:
[0026] Figure 1 It is the synthetic pathway for catalyzing L-tryptophan to 5-hydroxytryptophan constructed in the Escherichia coli engineering bacterium. L-TRP, L-tryptophan; GTP, guanosine triphosphate; NH 2 TP, neopterin dihydrotriphosphate; 6-BH 4 , 6-oxotetrahydropterin; BH 4 , tetrahydrobiopterin; 4α-OH-BH 4 , pterin-4α-methanolamine; qBH 2 , quinoid dihydropterin; Pyr, pyruvic acid; Indole, indole; GCHI, GTP cyclohydrolase I; PTPS, 6-oxotetrahydropterin synthase; SPR, sepiapterin reductase; PCD, pterin-4α-methanolamine dehydratase; DHPR, dihydropteridine reductase; TPH, tryptophan hydroxylase; TnaA, tryptophanase.
[0027] Figure 2 It is the map of the recombinant plasmid pEM-Spr-Dhpr-Ptps.
[0028] Figure 3It is the map of the recombinant plasmid pET28a-T5-TphI-GchI-Pcd. Specific implementation method:
[0029] 5-Hydroxytryptophan detection method (high performance liquid chromatography conditions):
[0030] Chromatographic column: C18 (5μm 4.6×250mm)
[0031] Mobile phase: 10% acetonitrile
[0032] Column temperature: 30°C
[0033] Detection wavelength: 276nm
[0034] Injection volume: 20μL
[0035] Flow rate: 1mL / min
[0036] Production intensity: Output ratio time
[0037] Example 1 Knockout of the tryptophanase gene tnaA in E. coli w3110
[0038] Designed according to the tnaA (tryptophanase gene) gene sequence in Escherichia coli W3110 in the NCBI database, using the CRISPR-ERA website ( designed tnaA at (http: / / crispr-era.stanford.edu / ) N20 sequence, and designing primers N20-tnaA and N20-A according to the N20 sequence. Then, using N20-tnaA and N20-A as primers and pTarget-F as the template, perform directional PCR amplification to obtain the linear vector of the pTarget-F-tnaA plasmid. After phosphorylating this linear vector, self-ligate. Transform the ligation product into JM109 competent cells, coat the LB plate with chiomycin resistance, pick the transformants, extract the plasmid, and verify by sequencing to obtain the plasmid pTarget-F-tnaA.
[0039] Designed according to the gene sequence of tnaA (tryptophanase gene) in Escherichia coli W3110 in the NCBI database, primers U-TnaA-S, U-TnaA-A, D-TnaA-S, and D-TnaA-A were designed. Then, using U-TnaA-S and U-TnaA-A as primers and the Escherichia coli W3110 genome as a template, the upstream fragment U-TnaA containing homologous arms was amplified; using D-TnaA-S and D-TnaA-A as primers and the Escherichia coli W3110 genome as a template, the upstream fragment D-TnaA containing homologous arms was amplified. Then, it was ligated to obtain the ΔTnaA fragment by one-step homologous recombination, and this fragment was further ligated to pMD19-T simple. The ligation product was transformed into JM109 competent cells, spread on an LB plate with ampicillin resistance, and transformants were picked. Plasmids were extracted and verified by sequencing to obtain the plasmid pMD19-T-ΔTnaA. Using U-TnaA-S and D-TnaA-A as primers and pMD19-T-ΔTnaA as a template, ΔTnaA-1 was obtained by PCR amplification.
[0040] ΔTnaA-1 and pTarget-F-tnaA were electrotransformed into the competent cells of E. coli w3110 containing the pCas9 plasmid. After rapid resuscitation in 1 mL of LB medium at 37 °C and 150 rpm for 1 h, it was spread on an LB solid medium plate containing both kanamycin and streptomycin. After inverted culture for 24 h, positive transformants were identified by colony PCR using the identification primers U-TnaA-2S and D-TnaA-A. Single colonies were picked, cultured with IPTG for 12 h, which could induce the sgRNA-pMB1 sequence to localize on ptarget and eliminate the pTarget-F-tnaA plasmid. Then it was spread on an LB solid medium plate containing kanamycin, and single colonies were selected to obtain the strain with the pTarget-F-tnaA plasmid eliminated. Single colonies were picked and inoculated into LB liquid medium. After overnight culture at 42 °C, single colonies that could grow on the non-resistant plate but not on the kanamycin-containing plate were screened and verified by colony PCR using the identification primers U-TnaA-2S and D-TnaA-A. The verified correct strain was named E. coli w3110ΔtnaA
[0041] The primer sequences are shown in Table 1.
[0042] Table 1 Primer sequences for PCR amplification of knockout fragments and overexpressed genes
[0043]
[0044] Example 2 Overexpression of Spr, Dhpr, and Ptps Genes
[0045] Using the artificially synthesized and codon-optimized Spr (as shown in SEQ ID NO.4), Dhpr (as shown in SEQ ID NO.5), and Ptps genes (as shown in SEQ ID NO.6) as templates, the homologous arm Spr fragment was amplified using primers Spr-A and Spr-S, the homologous arm Dhpr fragment was amplified using primers Dhpr-A and Dhpr-S, and the homologous arm Ptps fragment was amplified using primers Ptps-A and Ptps-S.
[0046] The primer sequences are shown in Table 1.
[0047] The amplified fragments were ligated by one-step homologous recombination to the pEM plasmid digested with Pst I and Xho I.
[0048] The above ligation product was transformed into JM109 competent cells, spread on an LB plate with ampicillin resistance, transformants were picked, plasmids were extracted, and sequencing verification was performed to obtain plasmid pEM-Spr-Dhpr-Ptps.
[0049] Example 3 Overexpression of Tph, GchI, and Pcd Genes
[0050] Using the artificially synthesized and codon-optimized Tph gene (as shown in SEQ ID NO.1), GchI gene (as shown in SEQ ID NO.2), and Pcd gene (as shown in SEQ ID NO.3) as templates, the homologous arm TphI fragment was amplified using primers TphI-A and TphI-S, the homologous arm GchI fragment was amplified using primers GchI-A and GchI-S, and the homologous arm Pcd fragment was amplified using primers Pcd-A and Pcd-S.
[0051] The primer sequences are shown in Table 1.
[0052] The amplified fragments were ligated by one-step homologous recombination to the pET28a-T5 plasmid digested with Hand III and Xho I.
[0053] The above ligation product was transformed into JM109 competent cells, spread on an LB plate with kanamycin resistance, transformants were picked, plasmids were extracted, and sequencing verification was performed to obtain plasmid pET28a-T5-Tph-GchI-Pcd.
[0054] Example 4 Construction of 5-Hydroxytryptophan Producing Strains
[0055] The obtained plasmids pEM-Spr-Dhpr-Ptps and pET28a-T5-TphI-GchI-Pcd were respectively transformed into the competent cells of E. coli w3110 and E. coli w3110ΔtnaA, and the plates with double resistance to kanamycin and ampicillin were coated for screening. The positive transformants obtained by colony PCR identification were the genetically engineered E. coli strains of the present invention, named TPH-1 (E. coli w3110 carrying pEM-Spr-Dhpr-Ptps and pET28a-T5-TphI-GchI-Pcd) and TPH-2 (E. coli w3110ΔtnaA pEM-Spr-Dhpr-Ptps and pET28a-T5-TphI-GchI-Pcd), respectively.
[0056] Example 5 Whole-cell catalysis of genetically engineered strains TPH-1 and TPH-2 for the synthesis of 5-hydroxytryptophan
[0057] The genetically engineered strains TPH-1 (as a control, without tnaA knockout) and TPH-2 were respectively cultured in 100 mL shaking flasks containing 20 mL of LB at a culture temperature of 37 °C and a rotation speed of 200 rpm. After 12 h of culture, they were inoculated into TB medium (50 mL / 250 mL Erlenmeyer flask) at an inoculation amount of 10% (V / V). When OD 600When it reached 0.6 - 0.8, 0.5 mM IPTG was added for induction, and at the same time, 10 g / L L-tryptophan was added as a substrate, and whole-cell catalysis was carried out at 30 °C for 48 h. After the reaction, centrifugation was performed at 4 °C and 8000 r / min for 10 min to collect the supernatant, and the conversion of L-tryptophan was detected by high-performance liquid chromatography. The genetically engineered strains TPH-1 and TPH-2 could consume 10 g / L L-tryptophan in 24 h, and the concentrations of 5-hydroxytryptophan synthesized were 3.52 g / L and 5.57 g / L respectively, the conversion rates were 35.2% and 55.7% respectively, and the production intensities were 0.073 g / L / h and 0.116 g / L / h respectively. It was reported in the literature that under the condition of adding 80 g / L whey powder and using 2 g / L tryptophan as a substrate, 1.302 g / L of 5-hydroxytryptophan could be synthesized, but its fermentation period was 72 h and the production intensity was only 0.018 g / L / h (Efficient synthesis of 5-hydroxytryptophanin Escherichia coli by bifunctional utilization of whey powder as a substratefor cell growth and inducer production. Journal of Biotechnology, 2024, 393, 100 - 108.).
Claims
1. A genetically engineered bacterium, characterized in that: The engineered bacteria include knocking out the tryptophanase gene tnaA, overexpressing The following genes: tryptophan hydroxylase gene TPH, tetrahydrobiopterin synthesis-related genes, pterin-4α-methanolamine dehydratase gene PCD and dihydropteridine reductase gene DHPR; genes related to tetrahydrobiopterin synthesis The genes for GTP cyclohydrolase I, GCHI, 6-pyruvyltetrahydrobiopterin synthase, PTPS, and bird butterfly SPR gene for succinate reductase.
2. The genetically engineered bacterium according to claim 1, characterized in that: The tryptophan hydroxylase gene TPH The nucleotide sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the GCHI gene is shown in SEQ ID NO.2 The nucleotide sequence of the PCD gene is shown in SEQ ID NO.3, and the nucleotide sequence of the SPR gene is shown in The nucleotide sequence of the DHPR gene is shown in SEQ ID NO.4, and the nucleotide sequence of the DHPR gene is shown in SEQ ID NO.5 As shown, the nucleotide sequence of the PTPS gene is shown in SEQ ID NO.
6.
3. The genetically engineered bacterium according to claim 1, characterized in that The tryptophanase gene tnaA is a nucleotide sequence shown in Sequence ID: CP062250.1 on KEGG.
4. A method for preparing the recombinant Escherichia coli according to claim 1, characterized in that: The following steps are involved: (1) The tryptophan hydroxylase genes TPH, GCHI and PCD were connected to the plasmid pET28a-T5 to construct the recombinant plasmid pET28a-T5-Tph-GchI-Pcd; (2) Connect the SPR, DHPR and PCD genes to the plasmid pEM to construct the recombinant plasmid pEM-Spr-Dhpr- Ptps; (3) The recombinant plasmid pET28a-T5-Tph-GchI-Pcd constructed in step (1) and the recombinant plasmid pEM-Spr-Dhpr-Ptps constructed in step (2) are transferred into the competent cell of Escherichia coli that does not express tnaA to obtain genetically engineered bacteria.
5. A method for whole-cell catalytic production of 5-hydroxytryptophan, characterized in that: The recombinant Escherichia coli according to any one of claims 1 to 3 is used to ferment L-tryptophan as a substrate to synthesize 5-hydroxytryptophan.
6. The method according to claim 5, characterized in that: The genetically engineered bacteria according to claim 1 are cultured in a 100 mL shake flask filled with 20 mL LB at a temperature of 37° C. and a rotation speed of 200 rpm. After culturing for 12 hours, TB culture medium is inoculated at a 10% inoculation amount V / V. When OD600 reaches 0.6 to 0.8, 0.5 mM IPTG is added for induction, and 10 g / L L-tryptophan is added as a substrate. Whole-cell catalysis is carried out at 30° C. for 48 hours. After the reaction is completed, centrifugation is carried out at 4° C. and 8000 r / min for 10 minutes, and the supernatant is collected, separated and purified to obtain 5-hydroxytryptophan.