Recombinant microorganism and application thereof in preparation of glycine
By constructing an enzyme combination of threonine aldolase, alcohol dehydrogenase, and isopropanol dehydrogenase and expressing it in microorganisms, the environmental protection and efficiency problems of glycine production in chemical synthesis methods were solved, and economical production of glycine through efficient conversion of threonine was achieved.
Patent Information
- Application Number
- CN202511306281.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2026-01-16
AI Technical Summary
Existing chemical synthesis methods for preparing glycine suffer from numerous byproducts, difficulty in catalyst recovery, difficulty in removing ammonium chloride impurities, and significant environmental pressures. Furthermore, microbial fermentation processes that do not primarily target glycine are not highly efficient.
An enzyme combination containing threonine aldolase, alcohol dehydrogenase, and isopropanol dehydrogenase was constructed and expressed in microorganisms. The threonine was converted into glycine through fermentation, thereby regenerating NAD+ and eliminating the need for additional PLP.
This method achieves efficient conversion of threonine to glycine without the addition of PLP and NAD+, improving conversion rate and economic efficiency while reducing production costs.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant microorganism and application thereof in preparation of glycine. BACKGROUND
[0002] As one of the smallest amino acids, glycine has been widely used in various fields due to its simple molecular structure and unique chemical properties. It is not only the basic unit of protein synthesis in living organisms, but also plays an important role in the food, pharmaceutical, and cosmetic industries. In the food industry, glycine is often used as a flavoring agent, flavor enhancer, and food preservative, which can effectively improve the taste and flavor of food and increase the deliciousness of food. In the pharmaceutical industry, glycine is widely used in the preparation of oral supplements and drugs for treating certain diseases, which has the function of promoting protein synthesis and repairing tissues. In the skin care industry, glycine is not only mild and non-irritating to the skin, but also helps to improve skin barrier function, moisturizing, and anti-inflammatory effects. In the agricultural field, glycine can promote the absorption of nitrogen by plants by cooperating with other nitrogen sources and plant nutrients, thereby improving the growth rate and yield of crops.
[0003] Most of the current synthesis methods use chemical synthesis methods such as alpha-chloroacetic acid or Strecker method to prepare glycine. Although chemical synthesis method is suitable for large-scale production, it has the disadvantages of difficult recovery of by-products and catalysts, more side reactions, and difficult removal of impurities such as ammonium chloride, and also has environmental pressure.
[0004] CN 116376995 B describes a method for preparing glycine and acetyl-CoA using threonine. Threonine is used as a substrate, and recombinant E. coli containing an aldolase gene and an acetylating acetaldehyde dehydrogenase gene is added for fermentation. During the fermentation process, threonine is decomposed into glycine and acetaldehyde, and acetaldehyde is directly converted into acetyl-CoA or acetaldehyde is converted into acetic acid and then into acetyl-CoA. However, this process route is not aimed at glycine as the main target product. SUMMARY
[0005] In order to solve the problems existing in the prior art, the present application provides a recombinant microorganism and application thereof in preparation of glycine.
[0006] In a first aspect, the present application provides an enzyme combination, which comprises: threonine aldolase, ethanol dehydrogenase, and isopropanol dehydrogenase; the threonine aldolase comprises an amino acid sequence as shown in SEQ ID NO. 1.
[0007] The amino acid sequence as shown in SEQ ID NO. 1 is: MTDQSQQFASDNYSGICPEAWAAMEKANHGHERAYGDDQWTARAADHFRKLFETDCEVFFAFNGTAANSLALSSLCQSYHSVICSETAHVETDECGAPEFFSNGSKLLTARSEGGKLTPASIREVALKRQDIHYPKPRVVTITQATEVGSVYRPDELKAISATCKELGLNLHMDGARFSNACAFLGCTPAELTWKAGIDVLCFGGTKNGMAVGEAILFFNRKLAEDFDYRCKQAGQLASKMRFLSAPWVGLLEDGAWLRHAAHANHCAQLLSSLVADIPGVELMFPVEANGVFLQMSEPALEALRNKGWRFYTFIGSGGARFMCSWDTEEARVRELAADIRAVMSA.
[0008] Further, the ethanol dehydrogenase comprises one of the following: (1) the amino acid sequence shown as SEQ ID NO. 2; (2) the amino acid sequence shown as SEQ ID NO. 2 with addition, substitution or deletion of one or more amino acids, and an amino acid sequence having the same function; and / or, The isopropanol dehydrogenase comprises one of the following: (3) the amino acid sequence shown as SEQ ID NO. 3; (4) the amino acid sequence shown as SEQ ID NO. 3 with addition, substitution or deletion of one or more amino acids, and an amino acid sequence having the same function.
[0009] The amino acid sequence shown as SEQ ID NO. 2: MSIPETQKGVIFYESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHAWHGDWPLPVKLPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGYTHDGSFQQYATADAVQAAHIPQGTDLAQVAPILCAGITVYKALKSANLMAGHWVAISGAAGGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRSIGGEVFIDFTKEKDIVGAVLKATDGGAHGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKSISIVGSYVGNRADTREALDFFARGLVKSPIKVVGLSTLPEIYEKMEKGQIVGRYVVDTSK.
[0010] the amino acid sequence as shown in SEQ ID NO. 3: MSTTGTTPATTGYAAEFAGRTALVTGAASGIGLATARRLGAGGARVVVADFNAEGAEKAAAELRAGGVEAAAVELDVTRPESVEAAVGFAVDTFGSLDLAVNNAGIGGPSAPTGEYDVAAYQRVVRTNLDGVFYSMRYELPAIEAAGKGGSIVNVASILGSVGFAGSPAYVAAKHGVVGLTKAAAAEYAARGIRINAVGPGFIDTPLLKTMDEAAYKGLVALHPAGRLGRSEEVAELIAFLLSDRASFVAGSYHLVDGAYTAV.
[0011] In a second aspect, the present application provides a nucleic acid molecule combination, which is used for encoding the enzyme combination as described above.
[0012] Further, the nucleic acid molecule for encoding the threonine aldolase comprises the nucleotide sequence as shown in SEQ ID NO. 4 or its complementary sequence; and / or, the nucleic acid molecule for encoding the alcohol dehydrogenase comprises one of the following: (1) the nucleotide sequence as shown in SEQ ID NO. 5; (2) the complementary sequence of the nucleotide sequence as shown in SEQ ID NO. 5; (3) a nucleotide sequence encoding the same functional protein as that of SEQ ID NO. 5 after adding, replacing, or deleting one or more nucleotides; and / or, A nucleic acid molecule for encoding isopropyl alcohol dehydrogenase includes one of the following: (1) a nucleotide sequence as shown in SEQ ID NO. 6; (2) a complementary sequence of the nucleotide sequence as shown in SEQ ID NO. 6; (3) a nucleotide sequence encoding the same functional protein as that of SEQ ID NO. 6 after adding, replacing, or deleting one or more nucleotides.
[0013] a nucleotide sequence as shown in SEQ ID NO. 4:
[0014] a nucleotide sequence as depicted in SEQ ID NO. 5:
[0015] a nucleotide sequence as shown in SEQ ID NO. 6: ATGAGCACCACCGGAACCACCCCCGCCACCACCGGGTACGCCGCCGAGTTCGCCGGCCGTACCGCCCTCGTCACCGGTGCCGCCTCCGGTATCGGCCTGGCCACCGCCCGCCGGCTCGGCGCCGGCGGCGCCCGGGTCGTCGTCGCCGACTTCAACGCCGAGGGCGCCGAGAAGGCCGCCGCCGAGCTGCGGGCCGGTGGCGTCGAGGCCGCCGCGGTCGAGCTGGACGTCACCCGTCCGGAGTCCGTCGAGGCGGCCGTCGGGTTCGCCGTCGACACGTTCGGCTCGCTGGACCTCGCCGTCAACAACGCCGGCATCGGCGGCCCCAGCGCCCCGACCGGCGAGTACGACGTGGCGGCCTACCAGCGCGTCGTGCGCACCAACCTCGACGGCGTCTTCTACTCGATGCGCTACGAACTGCCCGCCATCGAGGCGGCCGGCAAGGGCGGCTCGATCGTGAACGTCGCCTCCATCCTCGGCTCGGTCGGCTTCGCCGGCTCCCCCGCCTACGTCGCCGCCAAGCACGGCGTGGTCGGGCTGACGAAGGCGGCCGCCGCCGAGTACGCCGCCCGCGGCATCCGGATCAACGCGGTCGGTCCGGGCTTCATCGACACCCCCCTGCTCAAGACCATGGACGAGGCCGCCTACAAGGGGCTGGTCGCCCTGCACCCGGCCGGCCGCCTCGGGCGCTCCGAGGAGGTCGCGGAGCTGATCGTCTTCCTGCTGTCCGACCGCGCGTCCTTCGTCGCGGGCAGCTATCACCTGGTCGACGGCGCCTACACCGCCGTCTGA.
[0016] In a third aspect, the present application provides a biomaterial comprising the nucleic acid molecule composition as described above.
[0017] The biological material of the present application includes, but is not limited to, expression cassettes, vectors and other biological materials commonly used in microbial gene editing. The expression cassette of the present application includes a promoter, a coding sequence (corresponding to one or more nucleotide sequences in the aforementioned nucleic acid molecule combination) and a termination signal (such as a terminator and a polyadenylation signal) for terminating the transcription process, which can also guide the cell to add a Poly(A) tail at the end of the mRNA to increase the stability and translation efficiency of the mRNA. Commonly used are SV40 polyA, BGH polyA, etc. In addition, various elements can be added to the expression cassette, such as enhancers, introns, Kozak sequences, Shine-Dalgarno sequences or selection marker genes, etc. The vector of the present application includes, but is not limited to, expression vectors (extrachromosomal circular DNA molecules derived from bacteria or yeast), phage vectors or artificial chromosome vectors (bacterial artificial chromosome BAC or yeast artificial chromosome YAC), etc.
[0018] In a fourth aspect, the present application provides a recombinant microorganism, which comprises: the aforementioned enzyme combination, and / or the aforementioned nucleic acid molecule combination.
[0019] Further, the microorganism is a bacterium, preferably Escherichia coli, Bacillus subtilis or Corynebacterium glutamicum.
[0020] In some embodiments of the present application, the microorganism is Escherichia coli.
[0021] In a fifth aspect, the present application provides a kit, which comprises one or more of the following: the aforementioned enzyme combination, or the aforementioned nucleic acid molecule combination, or the aforementioned biological material, or the aforementioned recombinant microorganism.
[0022] In a sixth aspect, the present application provides the use of the aforementioned enzyme combination, or the aforementioned nucleic acid molecule combination, or the aforementioned biological material, or the aforementioned recombinant microorganism, or the aforementioned kit in the preparation of glycine.
[0023] Further, the use comprises: fermenting the recombinant microorganism, collecting the microbial cells after fermentation, and mixing the microbial cells, threonine and isopropyl alcohol for catalytic reaction; Preferably, the concentration of threonine is 20-70 g / L; and / or, The temperature of the catalytic reaction is 28-33°C.
[0024] In some specific embodiments, the initial concentration of microbial cells is 1-100 x 10 8 CFU / mL.
[0025] The recombinant microorganism constructed by the present application comprises a catalytic reaction system composed of threonine aldolase, ethanol dehydrogenase and isopropanol dehydrogenase, wherein the threonine aldolase decomposes threonine in the substrate into glycine and acetaldehyde (harmful product). Among them, acetaldehyde is converted into harmless ethanol under the catalysis of ethanol dehydrogenase, but this catalytic process needs to consume NADH, therefore, the present application expresses isopropanol dehydrogenase in the microorganism to regenerate NAD+, without additional addition of NAD+ in the reaction system, and meanwhile, the obtained acetone does not have a significant influence on the overall catalytic reaction system.
[0026] In addition, it is found in the research process of the present application that for the aforementioned specific threonine aldolase, the whole cell catalytic reaction system (recombinant microorganism) composed of the same can also dispense with the additional addition of pyridoxal phosphate (PLP), and can complete the high-efficiency conversion by relying on the PLP of the recombinant microorganism itself. PLP plays a necessary role in the catalytic reaction of threonine aldolase, but it is relatively expensive, and the method provided by the present application dispenses with the additional addition of PLP, which has significant economic value.
[0027] The present application has the following beneficial effects: The present application converts threonine aldolase, ethanol dehydrogenase and isopropanol dehydrogenase into the microorganism, and the recombinant microorganism obtained by construction can be used to convert threonine to produce glycine, which not only can convert acetaldehyde into relatively more harmless ethanol, but also can regenerate NAD+, thereby dispensing with the additional addition of NAD+. In addition, the specific threonine aldolase used in the present application in the whole cell catalytic reaction system can achieve a better threonine conversion rate without the addition of PLP and NAD+, which has important significance and economic value. BRIEF DESCRIPTION OF DRAWINGS
[0028] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.
[0029] Figure 1 is a schematic diagram of the fermentation broth provided by example 1 of the present application; wherein the left graph is the fermentation broth, and the right graph is the detection result of OD600.
[0030] Figure 2 is the HPLC detection result of the glycine product provided by example 2 of the present application.
[0031] Figure 3 is the HPLC detection result of the glycine product provided by example 2 of the present application. 1 H NMR method detection. DETAILED DESCRIPTION
[0032] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in conjunction with the drawings in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.
[0033] In the following examples, the experimental methods are conventional methods in the art, for example, refer to the experimental manuals in the art, or follow the suggested conditions in the manufacturer's instructions, if not specifically mentioned.
[0034] In the following examples, the experimental materials and reagents are commercially available, if not specifically mentioned.
[0035] Example 1 The present embodiment provides a method for constructing a recombinant microorganism, comprising: 1. Synthesis of nucleic acid molecules.
[0036] The nucleotide sequences of threonine aldolase, ethanol dehydrogenase and isopropanol dehydrogenase are downloaded according to the accession number, and biosynthesis is performed (commissioned to a biological company). The amino acid sequence of threonine aldolase (WP_065760197.1) is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 4. The amino acid sequence of ethanol dehydrogenase (NP_014555.1) is shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 5. The amino acid sequence of isopropanol dehydrogenase (WP_003971780.1) is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 6. Cloned into the vector pET-28a(+) by the biosynthesis company, to obtain plasmids pET-TA, pET-ADH and pET-ScCR.
[0037] 2. Construction of a recombinant expression vector.
[0038] (1) Linearize pRSFDuet-1 and pCDFDuet-1 plasmids by PCR technology or enzyme digestion method, recover the vector fragment and perform dpn1 digestion. The present embodiment uses loop p1-p4 for linearization.
[0039] (2) Use specific primers to amplify the corresponding nucleic acids from plasmids pET-TA, pET-ADH and pET-ScCR, respectively, to obtain the target fragment.
[0040] (3) The recovered ADH and ScCR target fragments are connected to the multiple cloning sites 1 and 2 of the linearized pRSFDuet-1 vector by homologous recombination to obtain two plasmids, pRSFDuet-ADH-ScCR and pRSFDuet-ScCR-ADH. Similarly, the recovered TA target fragment is connected to the multiple cloning sites 1 and 2 of the linearized pCDFDuet-1 vector to obtain pCDFDuet-TA (MCS1) and pCDFDuet-TA (MCS2).
[0041] (4) The constructed pRSFDuet-ADH-ScCR and pRSFDuet-ScCR-ADH plasmids are combined with pCDFDuet-TA (MCS1) and pCDFDuet-TA (MCS2) plasmids to transform E. coli BL21 (DE3) for co-expression.
[0042] The sequences of the primer pairs used are as follows: Construction of pRSFDuet-ADH-ScCR: ADH-F: CACAGCCAGGATCCGAATTCGATGTCTATCCCAGAAACTCAAAAAGGTGTT, ADH-R: GCATTATGCGGCCGCAAGCTTTTATTTAGAAGTGTCAACAACGTATCTACCAAC.
[0043] Loop p1: CGAATTCGGATCCTGGCTGTG, Loop p2: AAGCTTGCGGCCGCATAATGC.
[0044] ScCR-F: GATATACATATGGCAGATCTCATGAGCACCACCGGAACC, ScCR-R: GGTTTCTTTACCAGACTCGAGTCAGACGGCGGTGTAGGC.
[0045] Loop p3: GAGATCTGCCATATGTATATC, Loop p4: CTCGAGTCTGGTAAAGAAACC.
[0046] Construction of pRSFDuet-ScCR-ADH: ScCR-F: CACAGCCAGGATCCGAATTCGATGAGCACCACCGGAACC, ScCR-R: GCATTATGCGGCCGCAAGCTTTCAGACGGCGGTGTAGGC.
[0047] ADH-F: GATATACATATGGCAGATCTCATGTCTATCCCAGAAACTCAAAAAGGTGTT, ADH-R: GGTTTCTTTACCAGACTCGAGTTATTTAGAAGTGTCAACAACGTATCTACCAAC.
[0048] Loops p1-p4 are the same as the foregoing.
[0049] Construct pCDFDuet-TA (MCS1): TA-F: CACAGCCAGGATCCGAATTCGACCGATCAGAGCCAGCAGTTT, TA-R: GCATTATGCGGCCGCAAGCTTTGCAGACATAACCGCACGAAT.
[0050] Loops p1 and p2 are the same as the foregoing.
[0051] Construct pCDFDuet-TA (MCS2): TA-F: GATATACATATGGCAGATCTCACCGATCAGAGCCAGCAGTTT, TA-R: GGTTTCTTTACCAGACTCGAGTGCAGACATAACCGCACGAAT.
[0052] Loops p3 and p4 are the same as the foregoing.
[0053] (5) After resistance screening and PCR verification, positive clones of pRSFDuet-ADH-ScCR+ pCDFDuet-TA (MCS1), pRSFDuet-ADH-ScCR+ pCDFDuet-TA (MCS2), and pRSFDuet-ScCR-ADH+ pCDFDuet-TA (MCS1) and pRSFDuet-ScCR-ADH+ pCDFDuet-TA (MCS2) were finally obtained. They are named recombinant bacteria A, recombinant bacteria B, recombinant bacteria C and recombinant bacteria D in turn. Among them, recombinant bacteria A has been preserved in the China Center for Type Culture Collection of Wuhan University, and the preservation date is August 4, 2025. The classification name is Escherichia coli, the name is CABIO-Gly-VIII, and the preservation number is CCTCC M 20251772.
[0054] Example 2 This example is directed to the functional verification of the three recombinant bacteria constructed in Example 1, including the following process: 1. Culture medium (1) Initial fermentation medium.
[0055] Table 1. Components of initial fermentation medium
[0056] Adjust the pH to 7.0.
[0057] (2) Feeding medium.
[0058] Table 2. Components of feeding medium
[0059] Note: Glucose and yeast powder or yeast paste cannot be sterilized together. The initial glucose in the fermentation medium can be calculated by the amount added to the feeding bottle.
[0060] Substitute ammonia and glucose for the original yeast powder peptone and glycerol, 2. Fermentation culture of the strain 2.1 For the four recombinant microorganisms constructed in Example 1, the following fermentation process is carried out: (1) First-stage seed: pick a single colony from the plate or take 10 μL of bacterial solution from the bacteria preservation tube in the -80℃ refrigerator, add to a shaking tube containing 5 mL of LB medium and 10 μL of KanR+10 μL of SmR, and incubate at 37℃ overnight for 12 h.
[0061] (2) Second-stage seed: take 1 mL of the overnight culture seed solution and transfer it to 100 mL of LB medium, and add 200 uL of KanR+200 μL of SmR, and incubate at 37℃ for 4-5 h as the second-stage seed solution.
[0062] Note: KanR (25 mg / mL), SmR (40 mg / mL), IPTG (0.5 M).
[0063] (3) Empty sterilization: sterilize the fermentation tank.
[0064] (4) Medium sterilization: fermentation medium: sterilize at 121℃ for 20 minutes. Glucose solution: sterilize separately at 115℃ for 15 minutes. Water: take another empty bottle, add 320 mL of water, and sterilize together with the glucose solution under the same conditions (115℃, 15 minutes). This sterile water is used for subsequent preparation of 20% ammonia water.
[0065] 2.1 Fermentation control as follows: (1) Add glucose into the fermentor, the amount is 15 g / L. Seed liquid is added into the fermentor, the inoculation amount is 10%. At the same time, add the membrane filtered antibiotics KanR (6 mL) and SmR (3.75 mL).
[0066] (2) Culture conditions: temperature 37 ℃, air flow 2-6 L / min, DO control above 20%, rotation speed control 200 rpm-800 rpm, manual control according to the dissolved oxygen.
[0067] (3) Culture for about 5 h, OD is about 10, then start induction, add 3 mL IPTG. Monitor the glucose content of the fermentation system, control the concentration at 3-5 g / L.
[0068] (4) Observe the dissolved oxygen level, and when the sugar content is less than 3 g / L, start glucose feeding, the initial feeding rate can be 12 mL / h, and the rate can be adjusted later, and the remaining glucose is fed in 16-24 h. Ammonia (alkali pump) is used to adjust the pH and provide nitrogen source.
[0069] (5) Centrifuge to collect the bacteria.
[0070] The fermentation broth obtained after fermentation is shown in the left graph of Figure 1 , and the OD600 detection result is shown in the right graph of Figure 1 . The bacterial cells are collected by centrifugation, filtered and washed to remove the culture medium and metabolite residues to obtain microbial cells.
[0071] 3. Production of glycine 3.1 Preparation of reaction system.
[0072] Reaction system 1 is reacted in a reaction kettle, the reaction system is 1 L, including: threonine 45 g, isopropyl alcohol 38 mL, the microbial cells obtained by fermentation and centrifugation, the final concentration in the reaction system is 2.5×10 8 CFU / mL, and the rest is pure water.
[0073] Reaction system 2 is based on reaction system 1, and 0.1 mM of PLP and 0.1 mM of NAD+ are added to the catalytic reaction system.
[0074] Reaction system 3 is based on reaction system 1, and 0.1 mM of PLP and 0.25 mM of NAD+ are added to the catalytic reaction system.
[0075] 3.2 Reaction conditions Catalyze at 30 ℃ for 36 hours.
[0076] 3.3 Purification (1) After the catalytic reaction is completed, heat at 80°C for 10 min, and let stand for a period of time to precipitate most of the cells.
[0077] (2) Absorb the supernatant, and concentrate to about 10 L by rotary evaporation.
[0078] (3) Centrifuge the concentrated catalytic liquid to obtain a high-concentration glycine supernatant.
[0079] (4) After the supersaturated solution is formed, crystallization is performed.
[0080] (5) Filter, collect the crystalline crystals, and according to the crystallization condition, pass the filtered crystalline liquid through multiple times of supersaturated solution formation-crystallization to collect the crystals.
[0081] (6) Redissolve the collected crystals multiple times with pure water, and perform nanofiltration (150-300 Da).
[0082] (7) Re-concentrate the nanofiltered liquid to about 300 g / L, and perform low-temperature crystallization.
[0083] (8) Detect the purity of the glycine crystals, and if the purity reaches 99.9% and the transmittance is 98%, the product is considered to be qualified.
[0084] 3.4 Detection method The purity of the glycine product is detected by HPLC external standard method, 1 H NMR, and single crystal diffraction.
[0085] (1) HPLC method detection.
[0086] Take 1 g of glycine product and 1 g of glycine standard (purity 99%) and dry them in an oven at 60°C (about 12 h) to a constant weight. After each 100 mg of glycine sample and standard is taken and the solution is prepared, the FmocL is used for derivatization, and the concentration in the glycine feed liquid and the purity of the finished product are detected by the HPLC method.
[0087] The derivatization method includes: i) Dilute the sample to 10 mM.
[0088] ii) Take 50 μL of the sample, and add 250 μL of borate buffer (100 mM, pH 9.0) thereto.
[0089] iii) Add 300 μL of FmocCl.
[0090] iv) Shake for 10 s, and stand for 30 min.
[0091] v) Quench by adding 600 μL of 25% acetonitrile-boric acid solution (250 mM, pH 5.5).
[0092] vi) After filtration with 0.22 μm organic membrane, used for HPLC detection.
[0093] Liquid phase detection method is as follows: Gradient elution was used, instrument: SHIMADZU, column using C18 column (Agilent 5HC-C18 250 4.6mm), detection wavelength 254nm, column oven temperature 25℃, injection volume 10 μL. Mobile phase A: water containing 0.1% v / v trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% v / v trifluoroacetic acid.
[0094] Table 3 Gradient elution procedure
[0095] (2) 1 H NMR method detection.
[0096] 20 mg of glycine product was weighed and sent for detection, 1 H-NMR (400 MHz, OD2): δ / ppm 3.52 (2H, s). The results are shown, the purity of glycine product is high, and no other impurity peak appears. Figure 3
[0097] (3) Threonine conversion rate and glycine yield calculation.
[0098] Threonine conversion rate was measured by TLC thin layer chromatography by scanning the chromatographic spots (developing agent was ethanol: ammonia (9:1)).
[0099] Glycine yield was measured by HPLC to obtain the concentration of glycine in the catalyst solution x volume compared with the theoretical mass.
[0100] 4、Results.
[0101] Table 4 Threonine conversion rate and glycine catalytic yield results of recombinant bacteria A
[0102] Table 5 Threonine conversion rate and glycine catalytic yield results of recombinant bacteria B
[0103] Table 6 Threonine conversion rate and glycine yield results of recombinant bacteria C
[0104] Table 7 Threonine conversion rate and glycine yield results of recombinant bacteria D
[0105] The detection results of the purified sample of recombinant bacteria A and recombinant bacteria B in reaction system 1 are shown in Table 1. Figure 2 As shown in Table 1, the retention time of glycine is 5.3 minutes, and the purity of the synthesized glycine product is >99%.
[0106] The detection results of the purified sample of recombinant bacteria A in reaction system 1 by H NMR method are shown in Table 2. Figure 3 As shown in Table 2, the purity of the glycine product is high, and no other impurity peaks are present, and the peak area integral value ratio at the target chemical shift is >99%.
[0107] The above results show that the whole-cell catalytic system of the recombinant bacteria A-D constructed by the present application can completely eliminate the additional addition of the two expensive coenzyme factors PLP and NAD+, and there is no significant difference in the conversion rate and glycine yield, that is, the recombinant bacteria A-D can successfully guide the intracellular catalytic metabolism by relying on the coenzyme factors produced by themselves.
[0108] Experimental Example The present application further provides the experimental results of each control group used in the research process, which are specifically as follows: 1. Control group 1.
[0109] The threonine aldolase in Example 1 is replaced with the threonine aldolase with accession number WP_069998496.1, and the rest of the process is unchanged to obtain recombinant bacteria E, which is detected by the method in Example 2.
[0110] 2. Control group 2.
[0111] The threonine aldolase in Example 1 is replaced with the threonine aldolase with accession number WP_011027566.1, and the rest of the process is unchanged to obtain recombinant bacteria F, which is detected by the method in Example 2.
[0112] 3. Control group 3.
[0113] The threonine aldolase in Example 1 is replaced with the threonine aldolase with accession number WP_003105702.1, and the rest of the process is unchanged to obtain recombinant bacteria G, which is detected by the method in Example 2.
[0114] 4. Experimental results The results are shown in the following table. In the case of using other threonine aldolases, PLP (an expensive cofactor) needs to be added to the system, otherwise the threonine conversion rate will decrease significantly. However, the use of the threonine aldolase of the present application can significantly improve the technical effect without the use of PLP, which shows that the catalytic system constructed based on the threonine aldolase selected in the present application can be highly adaptive to the metabolic flow of the chassis strain.
[0115] Table 7 Threonine conversion rate and glycine yield results of recombinant bacteria E
[0116] Table 8 Threonine conversion and glycine yield results for recombinant bacteria F
[0117] Table 9 Threonine conversion and glycine yield results for recombinant bacteria G
[0118] From the above results, it can be seen that for recombinant bacteria E and recombinant bacteria G, the threonine conversion and glycine yield significantly decreased when the PLP and NAD+ concentrations were reduced. For recombinant bacteria F, the threonine conversion and glycine yield significantly decreased when no PLP and NAD+ were added.
[0119] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art will understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims
1. An enzyme combination, characterized in that, The enzyme combination comprises: threonine aldolase, ethanol dehydrogenase and isopropanol dehydrogenase; the threonine aldolase comprises an amino acid sequence as shown in SEQ ID NO.
1.
2. The enzyme combination according to claim 1, characterized in that, The ethanol dehydrogenase comprises one of the following: (1) an amino acid sequence as shown in SEQ ID NO. 2; (2) an amino acid sequence obtained by adding, replacing or deleting one or more amino acids of the amino acid sequence as shown in SEQ ID NO. 2, and an amino acid sequence having the same function; and / or, The isopropanol dehydrogenase comprises one of the following: (3) an amino acid sequence as shown in SEQ ID NO. 3; (4) an amino acid sequence obtained by adding, replacing or deleting one or more amino acids of the amino acid sequence as shown in SEQ ID NO. 3, and an amino acid sequence having the same function.
3. A combination of nucleic acid molecules, wherein, The nucleic acid molecule combination is used for encoding the enzyme combination of claim 1 or 2.
4. The combination of nucleic acid molecules according to claim 3, wherein The nucleic acid molecule for encoding the threonine aldolase comprises a nucleotide sequence as shown in SEQ ID NO. 4 or a complementary sequence thereof; and / or, The nucleic acid molecule for encoding the ethanol dehydrogenase comprises one of the following: (1) a nucleotide sequence as shown in SEQ ID NO. 5; (2) a complementary sequence of the nucleotide sequence as shown in SEQ ID NO. 5; (3) a nucleotide sequence obtained by adding, replacing or deleting one or more nucleotide sequences of the nucleotide sequence as shown in SEQ ID NO. 5, and a nucleotide sequence encoding a protein having the same function; and / or, The nucleic acid molecule for encoding the isopropanol dehydrogenase comprises one of the following: (1) a nucleotide sequence as shown in SEQ ID NO. 6; (2) a complementary sequence of the nucleotide sequence as shown in SEQ ID NO. 6; (3) a nucleotide sequence obtained by adding, replacing or deleting one or more nucleotide sequences of the nucleotide sequence as shown in SEQ ID NO. 6, and a nucleotide sequence encoding a protein having the same function.
5. A biomaterial, characterized by, The nucleic acid molecule combination of claim 3 or 4; the biological material is an expression cassette, a vector, a cell or a recombinant virus particle.
6. The biomaterial of claim 5, wherein, The biological material is a vector, comprising: vector 1 and vector 2; The vector 1 comprises: a nucleic acid molecule for encoding the threonine aldolase mentioned in the enzyme combination of claim 1; The vector 2 comprises: a nucleic acid molecule for encoding the ethanol dehydrogenase and the isopropanol dehydrogenase mentioned in the enzyme combination of claim 1; wherein the replicons of the vector 1 and the vector 2 are incompatible; Preferably, the vector skeletons of the vector 1 and the vector 2 are respectively selected from one of pRSFDuet and pCDFDuet; Further preferably, the skeleton of the vector 1 is pCDFDuet, and the skeleton of the vector 2 is pRSFDuet.
7. A recombinant microorganism, characterized in that, The recombinant microorganism comprises: The enzyme combination of claim 1 or 2; and / or, The nucleic acid molecule combination of claim 3 or 4; and / or, The biological material of claim 5 or 6.
8. The recombinant microorganism of claim 7, wherein, The recombinant microorganism is a bacterium; Preferably, the chassis strain of the recombinant microorganism is Escherichia coli, Bacillus subtilis or Corynebacterium glutamicum; Further preferably, the chassis strain of the recombinant microorganism is Escherichia coli BL21.
9. Use of the enzyme combination of claim 1 or 2, or the nucleic acid molecule combination of claim 3 or 4, the biological material of claim 5 or 6, or the recombinant microorganism of claim 7 or 8 in the preparation of glycine.
10. Use according to claim 9, characterized in that, The use comprises: fermenting the recombinant microorganism, collecting the microbial cells after fermentation, and mixing the microbial cells, threonine and isopropyl alcohol for catalytic reaction; Preferably, the concentration of the threonine is 20-70 g / L; and / or, The temperature of the catalytic reaction is 28-33℃.