A method for preparing D-proline by a biological conjugation chemistry method

Abstract

The present invention belongs to the field of the combination of biotechnology and chemical engineering, and relates to a method for preparing D-proline by a biological combination chemical method. By realizing the co-expression of glutamate racemase and D-glutamate cyclase in an engineered bacterium, using this engineered strain as a whole-cell catalyst, and using L-glutamate as a substrate, the high-efficient synthesis of D-proline is achieved by combining chemical methods. The application of the genetically engineered recombinant strain of the present invention in combination with chemical methods to the synthesis of D-proline has simpler raw material sources, lower costs, higher product yields, and better chiral purity compared with the prior art.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and chemical engineering, and relates to a method for preparing D-proline by a bio-conjugation chemistry method. More specifically, it relates to the construction of a genetically engineered recombinant strain E.coli BL21 / pETDuet-RacE-Dglucy, and a method for catalyzing the product thereof to combine with chemical synthesis of D-proline. Background Art

[0002] D-proline (molecular formula: C5H9NO2, CAS No.: 344-2532) is an important chiral reagent and chiral intermediate. It can be used as a resolution reagent and chiral reagent, and can also be used as a chiral intermediate for synthesizing some chiral drugs. For example, it can be used as a key chiral intermediate for the anti-migraine drug eletriptan. Eletriptan was developed by Pfizer and was first launched in Australia in 2001, and then launched in many countries. The market demand is relatively large. Its principle of action is mainly to inhibit the release of neuropeptides by activating 5-HT1-related receptors in the human body, contract intracranial blood vessels and inhibit neurogenic inflammation to exert an anti-migraine effect; in addition, D-proline is also a key precursor for synthesizing the phyllanthus alkaloid (-)-securinine A, which is used for the clinical treatment of diseases such as poliomyelitis, amyotrophic lateral sclerosis and chronic aplastic anemia; and D-proline is also a key precursor for synthesizing (R)-Harmicine, which is a β-carboline alkaloid with multiple pharmacological activities. And as an asymmetric organic catalyst, it is because D-proline has a rigid conformation and can catalyze multiple reactions such as asymmetric Mannich reaction, aldol reaction, Mannich–AzaMichael reaction, Morita-Bayllis-Hillman reaction, Heck cross-coupling reaction, multi-component reaction for synthesizing chiral molecules with various structures. In summary, D-proline has important value whether as a chiral reagent or as a pharmaceutical intermediate, and has broad market prospects.

[0003] At present, the preparation methods of D-proline can be divided into two types: chemical method and biological method. In the chemical method, industrially, it is mainly prepared by using chemical asymmetric transformation technology. This technology uses L-proline as the raw material, n-butanal as the catalyst, and reacts with L-tartaric acid in n-butyric acid solvent to prepare D-proline-L-tartrate, and then treats it with ammonia water in methanol to obtain D-proline. This method mainly has problems such as high production cost and large environmental pollution. Another chemical method for preparing D-proline uses pyrrolidine-2-carboxaldehyde as the raw material, asymmetrically reduces it to D-prolinol, and then oxidizes it to D-proline. However, this method requires the use of expensive raw materials and chiral metal catalysts, and the enantiomeric excess (ee) value of D-proline is not high either.

[0004] The methods for preparing D-proline by biological methods mainly include biological asymmetric synthesis method, biological resolution method, and enantioselective degradation method for preparing D-proline. Among them, the biological asymmetric synthesis method mainly uses L-arginine as the raw material, chemically synthesizes L-Cl-arginine, uses microbial cells to hydrolyze it into (S)-5-amino-2-chloropentanoic acid, and then automatically reverses and cyclizes to D-proline. This route has a very high cost and is not suitable for industrial application. The biological resolution method mainly uses a specific enzyme to act on the L-proline derivative in the DL-proline derivative, while the D-proline derivative is retained. The L-proline derivative is reduced to L-proline by enzymatic catalysis, and the separated L-proline can be used as the raw material for preparing DL-proline. The enantioselective degradation method for preparation mainly uses DL-proline as the raw material, and degrades the L-proline in DL-proline while a specific microorganism grows in the culture medium, and D-proline is retained. The advantage of this method is less environmental pollution, but the production yield is low, it depends on the substrate DL-proline raw material, the cost of the prepared D-proline is high, and it cannot be industrially applied.

[0005] Due to the technical and cost problems of preparing D-proline by biological methods, the existing technology in the market is chemical production. Therefore, aiming at the key problems of the existing chemical and biological methods for preparing D-proline, developing a more efficient and low-cost D-proline synthesis process technology has important value and significance. Summary of the Invention

[0006] The present invention provides a route for synthesizing D-proline by biological enzyme catalysis + chemical method. Compared with single chemical method and biological catalysis method, it selects the cheaper and more easily available substrate L-glutamic acid as the precursor substance, is not restricted by the source of raw materials, the enzymatic catalysis step has a complete reaction and a higher yield, the chemical method has a complete reaction and a higher chiral purity of the product, and it is a brand-new D-proline synthesis route.

[0007] To achieve this purpose, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing D-proline by a biological conjugation chemical method, comprising the following steps:

[0009] 1) Using a genetically engineered bacterium containing a co-expression vector of a glutamate racemase gene and a D-glutamate cyclase gene as a catalyst, and using L-glutamate as a precursor to catalytically synthesize 5-oxo-D-proline, a D-proline precursor;

[0010] 2) Chemically synthesizing D-proline from 5-oxo-D-proline.

[0011] For the method described above,

[0012] The glutamate racemase gene and the D-glutamate cyclase gene are constructed into an expression vector; the co-expression vector is transformed into an expression strain, and after antibiotic screening, it is an engineered recombinant strain containing the glutamate racemase and D-glutamate cyclase genes.

[0013] Furthermore,

[0014] The glutamate racemase gene and the D-glutamate cyclase gene are constructed into the Escherichia coli prokaryotic expression vector pETDuet-1. Among them, the glutamate racemase gene is constructed into the multiple cloning site MCS1 in pETDuet-1. The 5' end of the gene has an NcoI restriction site, and the 3' end of the gene has a HindIII restriction site; the D-glutamate cyclase gene is constructed into the multiple cloning site MCS2 of pETDuet-1. The 5' end of the gene has an NdeI restriction site, and the 3' end of the gene has an XhoI restriction site; the co-expression plasmid is constructed into the Escherichia coli expression strain BL21(DE3), and after antibiotic screening, it is the engineered recombinant strain E. coli BL21 / pETDuet-RacE-Dglucy containing the glutamate racemase and D-glutamate cyclase genes.

[0015] Even further, the amino acid sequence of the glutamate racemase gene RacE is as shown in SEQ ID NO.1; the amino acid sequence of the D-glutamate cyclase gene Dglucy is as shown in SEQ ID NO.2.

[0016] For the method described above,

[0017] The fermentation process of the engineered bacterium is as follows: Pick a single colony from the solid LB medium and inoculate it into the liquid LB medium. Incubate it overnight with shaking at 180 - 220 rpm and 30 - 37 °C. The next day, inoculate it into the TB fermentation medium at an inoculation amount of 0.5 - 2%, and incubate it with shaking at 180 - 220 rpm and 30 - 37 °C for 4 - 6 h. Wait until OD 600nmWhen = 0.4 - 0.6, lactose with a final concentration of 0.5 - 1% is added, and induction culture is carried out at 180 - 220 rpm and 25 - 30 °C for 8 - 12 h. The thalli are collected for subsequent whole - cell catalytic synthesis reaction.

[0018] In the method described above, the process of catalytically synthesizing the D - proline precursor 5 - oxo - D - proline:

[0019] After L - glutamic acid is dissolved in an appropriate amount of buffer, pyridoxal phosphate and MnCl2 are added. Then, after adding an appropriate amount of water and stirring to dissolve, the pH is adjusted, and a genetically engineered recombinant strain is added for reaction.

[0020] Furthermore,

[0021] In the catalytic synthesis system, the concentration of L - glutamic acid is 3.5 - 50 g / L, the reaction pH is 7.0 - 8.0, the reaction temperature is 30 - 35 °C, the rotation speed is 180 - 220 r / min, the content of pyridoxal phosphate is 20 - 50.0 mg / L, the content of MnCl2 is 20 - 50 mg / L, and the concentration of wet thalli is 200 - 400 g / L; the reaction time is 3 - 7 h.

[0022] In the method described above, 5 - oxo - D - proline finally obtains D - proline through three steps of esterification, acyl chlorination, and hydrolysis reactions.

[0023] Further comprising:

[0024] After the reaction solution in step 1) is ultrafiltered to remove the enzyme solution, a methanol solution is added for esterification reaction. 98% sulfuric acid is slowly added dropwise under stirring, and the reaction is carried out at 65 - 70 °C until the reaction solution becomes completely clear. Then it is cooled to 5 - 10 °C at low temperature, the pH is adjusted to 7.5 - 8.0, and the precipitate is collected; after the precipitate is dissolved, it is subjected to acyl chlorination reaction with phosgene. After the reaction is completed, a hydrochloric acid solution is added to the system, and then it is crystallized from alcohol to obtain the target product D - proline.

[0025] The present invention also provides an engineered recombinant strain. The glutamate racemase gene and D - glutamate cyclase gene are constructed into an expression vector; the co - expression vector is transformed into an expression strain, and after antibiotic screening, it is an engineered recombinant strain containing the glutamate racemase and D - glutamate cyclase genes;

[0026] Furthermore:

[0027] The glutamate racemase gene and D-glutamate cyclase gene were constructed into the prokaryotic expression vector pETDuet-1 of Escherichia coli. Among them, the glutamate racemase gene was constructed into the multiple cloning site MCS1 in pETDuet-1. The 5' end of the gene carried an NcoI restriction site, and the 3' end of the gene carried a HindIII restriction site. The D-glutamate cyclase gene was constructed into the multiple cloning site MCS2 of pETDuet-1. The 5' end of the gene carried an NdeI restriction site, and the 3' end of the gene carried an XhoI restriction site. The co-expression plasmid was constructed into the Escherichia coli expression strain BL21(DE3). After antibiotic screening, the engineered recombinant strain E. coli BL21 / pETDuet-RacE-Dglucy containing the glutamate racemase and D-glutamate cyclase genes was obtained.

[0028] Furthermore, the amino acid sequence of the glutamate racemase gene RacE is shown in SEQ ID NO.1; the amino acid sequence of the D-glutamate cyclase gene Dglucy is shown in SEQ ID NO.2.

[0029] The present invention also provides the application of the above-mentioned engineered recombinant strain in the preparation of D-proline.

[0030] The present invention provides the use of the recombinant strain for the catalytic preparation of 5-oxo-D-proline, a D-proline precursor. Among them, the glutamate racemase RacE catalyzes the synthesis of D-glutamate using L-glutamate as a substrate, and the D-glutamate cyclase Dglucy catalyzes the synthesis of 5-oxo-D-proline using D-glutamate as a substrate.

[0031] The present invention uses a method combining enzymatic method and chemical method to produce proline, abandoning the disadvantages of the chemical method for asymmetric transformation technology, utilizing the advantages of efficient synthesis of the enzymatic method, and selecting the bulk raw material glutamate as the starting substrate. The source of the raw material is simple and easy to obtain, and it is not restricted by the source of the raw material. Under the action of glutamate racemase and D-glutamate cyclase, 5-oxo-D-proline is generated, and then combined with chemical methods to generate D-proline. This technology has obvious advantages compared with the existing chemical synthesis method and biological method technologies: the raw materials are easy to obtain, the cost is lower, the racemization reaction of glutamate is combined with the cyclization reaction, the reaction occurs in the forward direction, the conversion efficiency is higher, and the chiral purity of the product is higher. It is a brand-new synthetic route for D-proline. Brief Description of the Drawings

[0032] Figure 1 It is a schematic diagram of the catalytic + chemical method for synthesizing D-proline by the genetic engineering recombinant strain of the present invention. Detailed Embodiments

[0033] The following examples are intended to further illustrate the present invention, rather than limiting the present invention.

[0034] Example 1: Construction of genetically engineered recombinant strains

[0035] The amino acid sequences of L-glutamate racemase (RacE accession number: WP_220220798.1) and D-glutamate cyclase (Dglucy accession number: NP_001347949.1) were downloaded from the NCBI database website, and codon optimization for E. coli expression was performed, and whole gene synthesis was performed (glutamate racemase gene: RacE (optimized nucleotide sequence is shown in SEQ ID NO. 3), D-glutamate cyclase gene: Dglucy (optimized nucleotide sequence is shown in SEQ ID NO. NO.4), after gene synthesis, it was constructed into the Escherichia coli prokaryotic expression vector pETDuet-1, wherein the glutamate racemase gene RacE was constructed into the multiple cloning site MCS1 in pETDuet-1 (Novagen), with an NcoI restriction site at the 5' end of the gene and a HindIII restriction site at the 3' end of the gene; the D-glutamate cyclase gene Dglucy was constructed into the multiple cloning site MCS2 of pETDuet-1, with an NdeI restriction site at the 5' end of the gene and an XhoI restriction site at the 3' end of the gene. After verification by enzyme digestion and sequencing, the co-expression plasmid was constructed into the Escherichia coli expression strain BL21 (DE3) using the CaCl2 heat shock transformation method, and an appropriate amount was applied to an LB solid plate containing ampicillin resistance, and cultured overnight at 37°C for 12-16h. The engineered recombinant strain E. coli containing the glutamate racemase and D-glutamate cyclase genes grew on the plate. BL21 / pETDuet-RacE-Dglucy.

[0036] Example 2: Fermentation of genetically engineered recombinant strains and expression of target protein

[0037] A single colony was picked from the LB solid culture medium and inoculated into the LB liquid culture medium, shaken at 200 rpm and 37°C overnight, and inoculated into the TB fermentation medium at a 2% inoculum the next day, shaken at 200 rpm and 37°C for 5-6 hours. When OD600nm = 0.6, lactose was added at a final concentration of 1%, and the culture was induced at 200 rpm and 25°C for 8-12 hours. The bacteria were collected and the subsequent whole-cell catalytic synthesis reaction was carried out using L-glutamate as the substrate.

[0038] Example 3: Catalytic reaction of genetically engineered recombinant strains

[0039] (1) Collect the cells. Transfer the fermented cells to a 50 mL centrifuge tube, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant.

[0040] (2) Resuspend in deionized water. Add appropriate amount of deionized water to the centrifuge tube and resuspend thoroughly with a pipette tip. Repeat 1-2 times.

[0041] (3) Weigh 3.5 g of L-glutamic acid. After dissolving it with a pH 7.5 phosphate buffer solution, weigh 5 mg of pyridoxal phosphate (PLP) and 5 mg of MnCl₂ into a reaction cup. Add 50 mL of water, stir to dissolve, adjust the pH to 7.00, add 20 mL of the genetically engineered recombinant strain, and make up the volume to 100 mL. The cell concentration after volume adjustment is 400 g / L. Reaction conditions: 30 °C, pH 7.50, shake culture at 180 - 220 r / min. At regular intervals, take samples and perform HPLC analysis of the product;

[0042] (4) Product detection method

[0043] HPLC detection conditions: Detection wavelength 210 nm, sample injection volume: 20 μL, flow rate: 1.0 mL / min, column temperature: room temperature, running time: 25 min;

[0044] Chromatographic column: Shim-pack GIST 4.6×150 mnm 5 μm;

[0045] Mobile phase: Phosphate solution (weigh 13.6 g of potassium dihydrogen phosphate and 2.2 g of sodium heptanesulfonate, dissolve in 1000 ml of water, adjust the pH to 2.5 with phosphoric acid, filter) - acetonitrile (950∶50). After filtering with a 0.45 μm aqueous filter membrane, degas for about 20 min and set aside.

[0046] Table 1: Synthesis of 5-oxo-D-proline

[0047]

[0048] Example 4: Synthesis of D-proline

[0049] The product 5-oxo-D-proline prepared by the method of Example 3 for 6 - 7 hours. After ultrafiltering the reaction solution to remove the enzyme solution, 100 mL of methanol solution was added for esterification reaction. 10 mL of 98% sulfuric acid was slowly added dropwise under stirring, and the temperature was maintained at 65 - 70 °C for 2 h until the reaction solution became completely clear. Then it was cooled to 5 - 10 °C at low temperature, and the pH was adjusted to 7.5 - 8.0 with 5 mol / L NaOH, and the precipitate was collected. After the precipitate was fully dissolved in dichloromethane, it was transferred to a three-necked flask with a cold trap, cooled to -10 °C, and then phosgene was introduced into the reaction flask with nitrogen for reaction. When all the phosgene was added, stirring was continued at -10 °C for 1 h, then the temperature was raised to room temperature and stirring was continued at room temperature for 12 h. Subsequently, the reaction was stopped, the solution and residual phosgene were dried with nitrogen, and the oil was obtained by vacuum concentration. The obtained oil was added with 50 mL of 3 mol / L hydrochloric acid and refluxed by heating for 12 h. After cooling, the mixture was concentrated by vacuum, then dissolved in water, and passed through a strong-acid cation exchange resin, and the product was eluted with water until the eluate was no longer acidic. After collecting the eluate and freeze-drying, D-proline was obtained.

[0050] Table 2: Comparison of the D-proline preparation method of the present invention with the existing methods

[0051]

[0052] [1] Zhang Xiaolin, Qi Jian, Tao Ying, Gao Yan, Cheng Jinxing. Process study on the preparation of D-proline by asymmetric conversion method [J]. Journal of Nanchang University (Engineering and Technology Edition), 2006(02): 119 - 121 + 133.

[0053] [2] Wu Fahao, Li Gang, Qu Songtao, etc. A method for synthesizing D-proline. China, CN 107827802 A [P]. 2017.

[0054] [3] Production of d-proline from l-arginine using Pseudomonas aeruginosa, Journal of Molecular Catalysis B: Enzymatic, Volume 6, Issue 3, 1999, Pages 359 - 367.

[0055] [4] Song S, Fung Kin Yuen V, Di L, Sun Q, Zhou K, Yan N. Integrating Biomass into the Organonitrogen Chemical Supply Chain: Production of Pyrrole and d-Proline from Furfural. Angew Chem Int Ed Engl. 2020 Nov 2;59(45):19846 - 19850.

[0056] [5] Zhang F F. Preparation of D - proline by microbial proline racemase - proline dehydrogenase cascade catalysis [D]. Chongqing University of Posts and Telecommunications, 2022.001175.

[0057] SEQ ID NO.1:

[0058] MDNRPIGVMDSGLGGLSVVRVIQQKLPNEEVIFVGDQGHFPYGTKDQAEVRQLALSIGAFLLKHDVKMMVV

[0059] ACNTATAAALPALQAALPIPVIGVIEPGARAALAQDKKGPIGVIATTATTTAGAYPATIERLAPGTPVIAKATQPMV

[0060] EIVEHGQTGTAKAQEVVSEQLMTFKEHPVKTLIMGCTHFPFLAPEISKAVGPTVALVDPAKETVATAKSWLEQH

[0061] QAMGNHAHPNYHLYSTGNLPDLRAGVNKWLLSGHFDLGTAQIEEGD

[0062] SEQ ID NO.2:

[0063] MTISFLLRSCLRSAVRSLPKAALIRNTSSMTEGLQPASVVVLPRSLAPAFESFCQGNRGPLPLLGQSEAVKTLPQLS

[0064] AVSDIRTICPQLQKYKFGTCTGILTSLEEHSEQLKEMVTFIIDCSFSIEEALEQAGIPRRDLTGPSHAGAYKTTVPCAT

[0065] IAGFCCPLVVTMRPIPKDKLERLLQATHAIRGQQGQPIHIGDPGLLGIEALSKPDYGSYVECRPEDVPVFWPSPLT

[0066] SLEAVISCKAPLAFASPPGCMVMVPKDTASSASCLTPEMVPEVHAISKDPLHYSIVSAPAAQKVRELESTIAVDPG

[0067] NRGIGHLLLKDELLQAALSLSHARSVLVTTGFPTHFNHEPPEETDGPPGAIALAAFLQALGKETAMVVDQRALN

[0068] LHMRIVEDAIRQGVLKTPIPILTYQGRSMEDARAFLCKDGDPKSPRFDHLVAIERAGRAADGNYYNARKMNIKH

[0069] LVDPIDDIFLAAQKIPGISSTGVGDGGNELGMGKVKAAVKKHIRNGDVIACDVEADFAVIAGVSNWGGYALAC

[0070] ALYILNSCQVHERYLRRATGPSRRAGEQSWIQALPSVAKEEKMLGILVENQVRSGVSGIVGMEVDGLPFHDVH

[0071] AEMIRKLVGATTVHM

[0072] SEQ ID NO.3:

[0073] ATGGATAACCGTCCGATTGGCGTTATGGATAGTGGCCTGGGTGGTCTGAGCGTTGTTCGTGTGATT

[0074] CAGCAGAAACTGCCGAATGAAGAAGTTATTTTTGTTGGCGATCAGGGTCATTTTCCGTATGGCACC

[0075] AAAGATCAGGCCGAAGTGCGCCAGCTGGCCCTGAGCATTGGCGCATTTCTGCTGAAACATGATGT

[0076] TAAAATGATGGTTGTGGCATGTAATACCGCAACCGCAGCCGCACTGCCGGCCTTACAGGCCGCTC

[0077] TGCCGATTCCGGTTATTGGTGTGATTGAACCGGGTGCCCGCGCCGCCCTGGCTCAGGACAAAAAA

[0078] GGTCCGATTGGCGTGATTGCAACCACCGCAACCACCACCGCAGGTGCCTATCCGGCAACCATTGA

[0079] ACGTCTGGCCCCGGGTACCCCGGTTATTGCAAAAGCAACCCAGCCGATGGTTGAAATTGTGGAAC

[0080] ATGGTCAGACCGGCACCGCAAAAGCCCAGGAAGTTGTTAGCGAACAGCTGATGACCTTTAAAGAA

[0081] CATCCGGTTAAAACCCTGATTATGGGTTGTACCCATTTTCCGTTTCTGGCCCCGGAAATTAGTAAAG

[0082] CAGTGGGTCCGACCGTTGCCCTGGTTGATCCGGCCAAAGAAACCGTGGCCACCGCAAAAAGCTGG

[0083] CTGGAACAGCATCAGGCAATGGGTAATCATGCCCATCCGAATTATCATCTGTATAGTACCGGTAAT

[0084] CTGCCGGATCTGCGTGCAGGCGTTAATAAGTGGCTGCTGAGTGGCCATTTTGATCTGGGTACCGC

[0085] ACAGATTGAAGAAGGTGAC

[0086] SEQ ID NO.4:

[0087] ATGACCATTAGCTTTCTGCTGCGCAGCTGTCTGCGCAGCGCAGTTCGTAGCCTGCCGAAAGCCGCACTGATT

[0088] CGCAATACCAGTAGCATGACCGAAGGTCTGCAGCCGGCCAGCGTTGTTGTGCTGCCGCGTAGTCTGGCACC

[0089] GGCATTTGAAAGTTTTTGTCAGGGTAATCGCGGTCCGCTGCCGCTGCTGGGTCAGAGTGAAGCCGTGAAAA

[0090] CCCTGCCGCAGCTGAGTGCCGTTAGTGATATTCGTACCATTTGCCCGCAGCTGCAGAAATATAAATTTGGTAC

[0091] ATGTACCGGTATCCTGACCAGCCTGGAAGAACATAGCGAACAGCTGAAAGAAATGGTGACCTTTATTATTGA

[0092] TTGCAGCTTTAGCATTGAGGAAGCCCTGGAACAGGCCGGTATTCCGCGTCGTGATCTGACCGGTCCGAGTC

[0093] ATGCAGGCGCCTATAAAACCACCGTGCCGTGTGCCACCATTGCAGGCTTTTGCTGCCCGCTGGTTGTGACCA

[0094] TGCGTCCGATTCCGAAAGATAAACTGGAACGCCTGCTGCAGGCAACCCATGCAATTCGCGGTCAGCAGGGT

[0095] CAGCCGATTCATATTGGCGATCCGGGCCTGCTGGGCATTGAAGCACTGAGCAAACCGGATTATGGCAGCTAT

[0096] GTTGAATGTCGCCCGGAAGATGTTCCGGTTTTCTGGCCGAGCCCGCTGACCAGCTTAGAAGCCGTTATTAGC

[0097] TGCAAAGCCCCGCTGGCATTTGCAAGTCCGCCGGGCTGTATGGTTATGGTTCCGAAAGATACCGCAAGCAG

[0098] TGCCAGCTGCCTGACCCCGGAAATGGTGCCGGAAGTTCATGCCATTAGCAAAGATCCGCTGCATTATAGCAT

[0099] TGTGAGCGCCCCGGCCGCACAGAAAGTGCGTGAACTGGAAAGCACCATTGCCGTGGATCCGGGTAATCGC

[0100] GGCATTGGTCATCTGCTGCTGAAAGATGAACTGCTGCAGGCCGCCCTGAGCCTGAGCCATGCCCGTAGTGT

[0101] GCTGGTGACCACCGGTTTTCCGACCCATTTTAATCATGAACCGCCGGAAGAAACCGATGGCCCGCCGGGTG

[0102] CAATTGCACTGGCAGCATTTCTGCAGGCACTGGGCAAAGAAACCGCAATGGTGGTTGATCAGCGTGCCCTG

[0103] AATCTGCACATGCGTATTGTGGAAGATGCCATTCGTCAGGGTGTTCTGAAAACCCCGATTCCGATTCTGACCT

[0104] ATCAGGGTCGCAGCATGGAAGATGCACGTGCATTTCTGTGTAAAGATGGCGATCCGAAAAGTCCGCGCTTT

[0105] GATCATCTGGTGGCCATTGAACGCGCAGGCCGTGCCGCAGATGGCAATTATTATAATGCACGCAAAATGAAT

[0106] ATCAAGCATCTGGTTGATCCGATTGATGATATTTTTCTGGCAGCCCAGAAAATTCCGGGTATTAGTAGTACCG

[0107] GTGTGGGTGACGGTGGCAATGAACTGGGTATGGGTAAAGTTAAAGCAGCAGTGAAAAAACATATCCGCAAT

[0108] GGTGACGTGATTGCATGTGATGTTGAAGCCGATTTTGCAGTTATTGCCGGCGTTAGTAATTGGGGCGGTTAT

[0109] GCACTGGCCTGTGCCCTGTATATTCTGAATAGTTGCCAGGTGCATGAACGCTATCTGCGCCGTGCAACCGGTC

[0110] CGAGCCGTCGTGCTGGCGAACAGAGCTGGATTCAGGCACTGCCGAGTGTGGCCAAAGAAGAAAAAATGCT

[0111] GGGTATTCTGGTGGAAAATCAGGTGCGTAGCGGCGTGAGTGGCATTGTGGGCATGGAAGTTGATGGCCTG

[0112] CCGTTTCATGATGTGCATGCCGAAATGATTCGTAAACTGGTTGGCGCAACCACCGTGCACATG

Claims

1. A method for preparing D-proline precursor 5-oxo-D-proline, characterized in that, It includes the following steps: Using a genetically engineered bacterium containing a co-expression vector of glutamate racemase gene and D-glutamate cyclase gene as a catalyst, with L-glutamate as a precursor, to catalytically synthesize 5-oxo-D-proline, the precursor of D-proline; Constructing the glutamate racemase gene and D-glutamate cyclase gene into an expression vector; transforming the co-expression vector into an expression strain, and after antibiotic screening, it is an engineered recombinant strain containing the glutamate racemase and D-glutamate cyclase genes; The amino acid sequence of the glutamate racemase gene RacE is as shown in SEQ ID NO.1; the amino acid sequence of the D-glutamate cyclase gene Dglucy is as shown in SEQ ID NO.2; The process of catalytically synthesizing 5-oxo-D-proline, the precursor of D-proline: After dissolving L-glutamate in an appropriate amount of buffer, adding pyridoxal phosphate and MnCl2, stirring to dissolve, adjusting the pH, and adding the above-mentioned engineered recombinant strain for reaction.

2. The method according to claim 1, wherein The glutamate racemase gene and the D-glutamate cyclase gene were constructed into the prokaryotic expression vector pETDuet-1 of Escherichia coli. Among them, the glutamate racemase gene was constructed into the multiple cloning site MCS1 in pETDuet-1. The 5' end of the gene carried an NcoI restriction site, and the 3' end of the gene carried a HindIII restriction site. The D-glutamate cyclase gene was constructed into the multiple cloning site MCS2 of pETDuet-1. The 5' end of the gene carried an NdeI restriction site, and the 3' end of the gene carried an XhoI restriction site. The co-expression plasmid was constructed into the Escherichia coli expression strain BL21(DE3). After antibiotic screening, it was the engineered recombinant strain containing the glutamate racemase and D-glutamate cyclase genes E.coli BL21 / pETDuet-RacE-Dglucy.

3. The method according to claim 1, wherein The fermentation process of the engineered bacteria is as follows: Pick a single colony from the LB solid medium and inoculate it into the LB liquid medium. Incubate it overnight with shaking at 180 - 220 rpm and 30 - 37°C. The next day, inoculate it into the TB fermentation medium at an inoculation amount of 0.5 - 2%, and incubate it with shaking at 180 - 220 rpm and 30 - 37°C for 4 - 6 h. When OD 600nm = 0.4 - 0.6, add lactose with a final concentration of 0.5 - 1%, and induce culture at 180 - 220 rpm and 25 - 30°C for 8 - 12 h. Collect the bacterial cells for subsequent whole-cell catalytic synthesis reaction.

4. The method according to claim 1, wherein In the catalytic synthesis system, the concentration of L-glutamate is 3.5 - 50 g / L, the reaction pH is 7.0 - 8.0, the reaction temperature is 30 - 35 °C, 180 - 220 r / min, the content of pyridoxal phosphate is 20 - 50 mg / L, the content of MnCl2 is 20 - 50 mg / L, and the wet cell concentration is 200 - 400 g / L; the reaction time is 3 - 7 h.

5. Engineered recombinant strain, the glutamate racemase gene and D-glutamate cyclase gene are constructed into an expression vector; transforming the co-expression vector into an expression strain, and after antibiotic screening, it is an engineered recombinant strain containing the glutamate racemase and D-glutamate cyclase genes; The glutamate racemase gene and D-glutamate cyclase gene were constructed into the Escherichia coli prokaryotic expression vector pETDuet-1. Among them, the glutamate racemase gene was constructed into the multiple cloning site MCS1 in pETDuet-1. The 5' end of the gene carried an NcoI restriction site, and the 3' end of the gene carried a HindIII restriction site. The D-glutamate cyclase gene was constructed into the multiple cloning site MCS2 of pETDuet-1. The 5' end of the gene carried an NdeI restriction site, and the 3' end of the gene carried an XhoI restriction site. The co-expression plasmid was constructed into the Escherichia coli expression strain BL21(DE3). After antibiotic screening, it was the engineered recombinant strain containing the glutamate racemase and D-glutamate cyclase genes E.coli BL21 / pETDuet-RacE-Dglucy; The amino acid sequence of the glutamate racemase gene RacE is as shown in SEQ ID NO.1; the amino acid sequence of the D-glutamate cyclase gene Dglucy is as shown in SEQ ID NO.2.

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