A D-p-hydroxyphenylglycine high-yielding strain and its application

Abstract

The invention discloses a D-p-hydroxyphenylglycine high-yield strain and its application. The invention improves the expression of D-p-hydroxyphenylglycine by connecting mutants of D-hydantoinase and D-carbamoylase to a double expression plasmid pETduet-1. The strain produces 30g / LD-p-hydroxyphenylglycine after 12h of transformation, and the yield reaches 24.9g / L, and the molar conversion rate reaches 95.4%. The strain produces 40g / LD-p-hydroxyphenylglycine after 12h of transformation, and the yield reaches 30.4g / L, and the molar conversion rate reaches 87.4%. The strain produces 50g / LD-p-hydroxyphenylglycine after 12h of transformation, and the yield reaches 29.8g / L, and the yield reaches 33.0g / L after 24h of transformation, and the molar conversion rate reaches 75.9%. The method of the invention has great potential and extensive value for industrial use in increasing the yield of D-p-hydroxyphenylglycine.

Description

Technical Field

[0001] The invention relates to a D-p-hydroxyphenylglycine high-yield strain and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] D-p-Hydroxyphenylglycine is an important drug and food additive. It is widely used in the industrial production of semi-synthetic antibiotics, peptides, hormones, pyrethroids, pesticides, sweeteners, etc. It is mainly used for semi-synthetic antibiotics such as amoxicillin, cefadroxil and cefoperazone. D-p-Hydroxyphenylglycine is the D-type amino acid with the largest demand at present, and its output reaches thousands of tons worldwide each year.

[0003] The preparation methods of D-p-hydroxyphenylglycine are mainly divided into chemical synthesis and hydantoinase conversion. At present, chemical synthesis is the main production method of D-p-hydroxyphenylglycine, mainly including: asymmetric transformation method, induced crystallization method and chemical resolution method. Although there are many chemical methods for synthesizing D-p-hydroxyphenylglycine, these methods have problems of harsh reaction conditions, large environmental pollution, low production efficiency and high production cost to varying degrees. Therefore, more and more researchers are seeking to use enzyme conversion to synthesize D-p-hydroxyphenylglycine. The current enzyme conversion method is mainly hydantoinase method: using D, L-p-hydroxyphenylhydantoin (D, LHPH) as a substrate, under the catalysis of D-hydantoinase (DHase) and D-carbamoylase (DCase), the intermediate N-carbamoyl D-p-hydroxyphenylglycine (D-CpHPG) is used to generate optically pure D-HPG, which is called the hydantoinase method for producing D-HPG.

[0004] However, in the process of hydantoin enzymatic production, D-carbamoylase is restricted in its industrial production due to problems such as expression and stability. Therefore, there are also biotransformation methods for producing D-p-hydroxyphenylglycine. Currently, biotransformation methods mainly include Escherichia coli transformation and Bacillus subtilis. Bacillus subtilis cannot solve the problems of low expression, stability and soluble expression of N-carbamoylase, and because it is a Gram-positive bacterium, its thick cell wall also becomes one of the limiting factors in the catalytic process. The existing Escherichia coli used for D-p-hydroxyphenylglycine production has a low expression level when expressing N-carbamoylase, resulting in the inability to further increase the yield of D-p-hydroxyphenylglycine. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for expressing D-hydantoinase (GsDH) and D-carbamoylase mutant (AkDC A273T) can achieve the purpose of increasing the production of D-p-hydroxyphenylglycine.

[0006] The first object of the present invention is to provide a high-yield strain of D-p-hydroxyphenylglycine, wherein the strain uses plasmid pETduet-1 as a vector to express D-hydantoinase (GsDH) and D-carbamoylase mutant (AkDC A273T ).

[0007] Furthermore, the D-carbamoylase mutant and D-hydantoinase are both expressed by T7 promoter, wherein the D-carbamoylase mutant is constructed behind the first promoter and the D-hydantoinase is constructed behind the second promoter.

[0008] Furthermore, the sequence of the recombinant plasmid constructed using plasmid pETduet-1 as a vector is shown in SEQ ID NO.7.

[0009] The D-hydantoinase (GsDH) of the present invention is derived from Geobacillus stearothermophilus, and the D-carbamoylase (AkDC) of the present invention is derived from Agrobacterium sp. (strain KNK712).

[0010] Furthermore, the amino acid sequence of the D-hydantoinase is shown in SEQ ID NO.1.

[0011] Furthermore, the amino acid sequence of the D-carbamoylase is shown in SEQ ID NO.3.

[0012] Furthermore, the amino acid sequence of the D-carbamoylase mutant is shown in SEQ ID NO.5.

[0013] Furthermore, the E. coli host is E. coli BL21 (DE3).

[0014] The second object of the present invention is to provide a method for constructing the D-p-hydroxyphenylglycine high-yielding strain, comprising the following steps:

[0015] (1) Obtaining gene fragments encoding D-carbamoylase mutant and D-hydantoinase (AkDC A273T and GsDH);

[0016] (2) The gene fragment encoding D-carbamoylase (AkDC A273T ) was connected to the pETduet-1 vector, in which the D-carbamoylase gene fragment (AkDC A273T) is located between the restriction sites BamHI and EcoRI. The resulting recombinant plasmid is pETduet-1-AkDC A273T ;

[0017] (3) Introducing the recombinant plasmid described in step (2) into E. coli BL21 (DE3), culturing and extracting the plasmid to verify and enrich the recombinant plasmid pETduet-1-AkDC A273T ;

[0018] (4) Connecting the gene fragment encoding D-hydantoinase (GsDH) to the recombinant plasmid pETduet-1-AkDC described in step (3) A273T The gene fragment of D-hydantoinase (GsDH) is located at the restriction site XhoⅠ. The resulting recombinant plasmid is pETduet-1-AkDC A273T -GsDH;

[0019] (5) Transforming the recombinant plasmid obtained in step (4) into E. coli BL21 (DE3) to obtain the D-p-hydroxyphenylglycine high-yielding strain.

[0020] Furthermore, the recombinant plasmid pETduet-1-AkDC A273T The nucleotide sequence of -GsDH is shown in SEQ ID NO.7.

[0021] The third object of the present invention is to provide the use of the D-p-hydroxyphenylglycine high-yielding strain in the production of D-p-hydroxyphenylglycine.

[0022] Furthermore, the application is to use the whole cells of the D-p-hydroxyphenylglycine high-yielding strain after fermentation as a catalyst to catalyze DL-p-hydroxyphenylglycine to produce D-p-hydroxyphenylglycine.

[0023] Furthermore, the fermentation of the D-p-hydroxyphenylglycine high-yielding strain is performed by inoculating the D-p-hydroxyphenylglycine high-yielding strain into a seed liquid culture medium to obtain a seed liquid, and then inoculating the seed liquid into a fermentation medium for cultivation.

[0024] Furthermore, the seed liquid culture medium includes: 8-12 g / L tryptone, 4-6 g / L yeast powder, and 8-12 g / L NaCl; the fermentation medium includes: 10-14 g / L tryptone, 22-26 g / L Angel yeast powder, and potassium dihydrogen phosphate (KH 2 PO 4 2.2~2.4g / L, potassium dihydrogen phosphate K 2 HPO 4 3H 2 O 16~17g / L, glycerol 3~5g / L.

[0025] Furthermore, the fermentation culture conditions are: temperature 35-38°C, rotation speed 200-250rpm, culture 2-3h, adding 0.3-0.5mmol / L IPTG for induction, induction temperature 22-26°C, time 10-20h and rotation speed 200-250rpm.

[0026] In the present invention, the protein verification method of the strain production is to take the above wet bacteria and add them into an EP tube, wash with a buffer solution, then add a buffer solution and mix, and then ultrasonically disrupt. After centrifugation, take the supernatant and the precipitate respectively, and perform SDS-PAGE polyacrylamide gel electrophoresis. The conditions of the ultrasonic disruption can be specifically: ultrasonic power 27%, ultrasonic 10min, ultrasonic 3s, interval 3s. The conditions of the centrifugation can be specifically: 12000rpm 5min.

[0027] Furthermore, the conditions of the catalytic reaction are 38-42° C., 180-250 rpm, and a reaction pH of 7.5-8.0.

[0028] The beneficial effects of the present invention are:

[0029] The invention improves the expression amount of N-carbamoylase by connecting D-hydantoinase derived from Geobacillus stearothermophilus and a mutant of D-carbamoylase derived from Agrobacterium sp. (strain KNK712) to a double expression plasmid pETduet-1. The strain produces 30g / L D-p-hydroxyphenylglycine at the shaking flask level after 12h transformation, and the yield reaches 24.9g / L, the molar conversion rate reaches 95.4%, and the yield reaches 25.4g / L after 24h transformation, and the molar conversion rate reaches 97.3%; the yield of 40g / L D-p-hydroxyphenylglycine after 12h transformation reaches 30.4g / L, the molar conversion rate reaches 87.4%, and the yield reaches 30.8g / L after 24h transformation, and the molar conversion rate reaches 88.5%; the yield of 50g / L D-p-hydroxyphenylglycine after 12h transformation reaches 29.8g / L, the molar conversion rate reaches 68.5%, and the yield reaches 33.0g / L after 24h transformation, and the molar conversion rate reaches 75.9%. The method of the present invention has great potential and extensive value in industrial use for increasing the yield of D-p-hydroxyphenylglycine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown is a schematic diagram of plasmid construction;

[0031] Figure 2The protein gel images of D-hydantoinase and D-carbamoylase in different vectors are shown. pACYCduet-GsDH-AkDC represents GsDH behind the first promoter and AkDC behind the second promoter, and the same applies to the others.

[0032] Figure 3 Shown are the transformation effects of 20 g / L substrate with different carriers;

[0033] Figure 4 Shown are the HPLC spectra of each standard;

[0034] Figure 5 Shown is the production of D-p-hydroxyphenylglycine at different time periods;

[0035] Figure 6 Shown are the yields of D-p-hydroxyphenylglycine produced at substrate concentrations of 30 g / L, 40 g / L and 50 g / L after conversion for 12 h and 24 h. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples are repeated three times, and the results are taken as average values.

[0038] The D-hydantoinase from Geobacillus stearothermophilus is shown in SEQ ID NO.1, and its encoding gene is shown in SEQ ID NO.2. The D-carbamoylase from Agrobacterium sp. (strain KNK712) is shown in SEQ ID NO.3, and its encoding gene is shown in SEQ ID NO.4. The D-carbamoylase mutant is shown in SEQ ID NO.5, and its encoding gene is shown in SEQ ID NO.6.

[0039] Example 1: Recombinant plasmid pETduet-1-AkDC A273T -Construction of GsDH

[0040] 1. Synthesize the double-stranded DNA molecule shown in Sequence 6 of the sequence table.

[0041] 2. Using the DNA molecule synthesized in step 1 as a template, PCR amplify AkDC using the primer pair consisting of P1 and P2.A273T fragment, gel recovery PCR product;

[0042] The PCR primers used to amplify the gene encoding D-carbamoylase (AkDC) were P1 and P2:

[0043] P1:5'-TCATCACCACAGCCA GGATCC GATGACCCGTCAGATGATTCTGG-3'

[0044] P2:5'-AGGCGCGCCGAGCTC GAATTC TTACAGTTCCGCAATCAGGCC-3'

[0045] 3. Use restriction endonucleases BamHⅠ and EcoRⅠ to double-digest the vector pETduet-1, and recover the digestion products by gel;

[0046] 4. Use homologous recombinase ExnaseⅡ to connect the gel recovery products of step 2 and step 3 to obtain the recombinant plasmid pETduet-1-AkDC A273T ;

[0047] 5. The recombinant plasmid obtained in step 4 was introduced into E. coli BL21 (DE3), cultured and extracted to verify and enrich the recombinant plasmid pETduet-1-AkDC A273T ;;

[0048] 6. Use restriction endonuclease XhoⅠ to perform single enzyme digestion on the recombinant plasmid enriched in step 5, and perform gel recovery on the digestion product;

[0049] 7. Synthesize the double-stranded DNA molecule shown in Sequence 2 of the sequence table.

[0050] 8. Using the DNA molecule synthesized in step 7 as a template, use the primer pair consisting of P3 and P4 to amplify the GsDH fragment by PCR, and recover the PCR product by gel;

[0051] The PCR primers used to amplify the gene encoding D-hydantoinase (GsDH) are P3 and P4:

[0052] P3:5'-GCTGACGTCGGTACCCTCGAGatgaccaaaattattaaaaatggtacca-3'

[0053] P4:5'-GGTTTCTTTTACCAGACTCGAGttaaatggtcagttcttcgctctg-3'

[0054] 9. Use homologous recombinase Exnase II to connect the gel recovery products of step 6 and step 8 to obtain the recombinant plasmid pETduet-1-AkDC A273T -GsDH.

[0055] The results of sequencing the correct pETduet-1-AkDC-GsDH were as follows: pETduet-1 was used as a vector, and the gene of D-carbamoyl hydrolase (AkDC A273T ), and the gene of D-hydantoinase (GsDH) shown in sequence 2 of the sequence list was inserted into the XhoⅠ restriction site. The plasmid construction map is shown in Figure 1 .

[0056] The obtained pETduet-1-AkDC A273T -GsDH plasmid was transformed into E. coli BL21 (DE3) to obtain recombinant E. coli 01.

[0057] Example 2: Shake flask fermentation and cell collection of recombinant E. coli 01 strain

[0058] Pick the recombinant strain of E.coli 01 for shake flask fermentation. The single clone was inoculated into 30mL (100mL shake flask) of LB medium containing 10mg / mL Amp as the seed liquid for shake flask fermentation, and cultured at 37°C, 200rpm for 7h. The seed liquid culture medium LB is: tryptone (TRYPTONE) 10g / L, yeast powder (YEAST EXTRACT) 5g / L, NaCl 10g / L, the inoculation amount of shake flask fermentation is 2%, the fermentation conditions are temperature 37°C, rotation speed 220rpm, and the initial pH is controlled at about 7.0. The fermentation medium is: tryptone 12g / L, Angel yeast powder 24g / L, potassium dihydrogen phosphate KH 2 PO 4 2.31g / L, potassium dihydrogen phosphate K 2 HPO 4 3H 2 O, glycerol 4g / L. After culturing for 2.5h, IPTG with a final concentration of 0.42mmol / L was added for induction, the induction temperature was 25℃, the induction time was 16h, and the rotation speed was 220rpm; after the induction, the wet cells were collected at 6000rpm for 12min and refrigerated at -40℃.

[0059] Example 3: Expression of target protein in recombinant strains

[0060] Take 0.05g of the above wet bacteria in a 1.5mL EP tube, add 1mL of pH 7.8 Tris-HCl buffer and mix, 12000rpm 1min, discard the supernatant, add 1mL of pH 7.8 Tris-HCl buffer and mix, ultrasonic disruption (ultrasonic power 27%, ultrasonic 10min, ultrasonic 3s, interval 2s), then centrifuge at 12000rpm for 5min, take 30uL of supernatant and add 10uL 4Xprotein loading buffer, mix, and load on SDS-PAGE electrophoresis. At the same time, discard the supernatant in the 1.5mL EP tube, add 1mL of pH 7.8 Tris-HCl buffer to mix and precipitate, take 30uL of suspension and add 10uL 4Xproteinloading buffer, mix, load on SDS-PAGE electrophoresis, and then run SDS-PAGE gel.

[0061] The results of polyacrylamide gel electrophoresis are shown in Figure 2 13 and 14, the molecular weight of D-hydantoinase (GsDH) is 54 kDa, and D-carbamoylase (AkDC A273T ) has a molecular weight of 34 kDa.

[0062] Example 4: Preparation of D-p-hydroxyphenylglycine by whole cells

[0063] In a 20mL small brown bottle, 0.08g of whole cell E.coli 01 after induction culture, 0.12g (30g / L), 0.16g (40g / L) and 0.20g (50g / L) DL-p-hydroxyphenylhydantoin (DL-HPH), and 4mL pH7.8 Tris-HCl buffer were added respectively, and the mixture was reacted in a constant temperature shaker at 40°C and 220rpm for 24h, and the reaction pH was 7.8. The reaction solution sample after conversion was centrifuged at 12000rpm for 5-10min, and the supernatant was diluted with ultrapure water, and after passing through a 0.22μm water film, high-performance liquid chromatography HPLC analysis was performed.

[0064] HPLC detection used a chromatographic column (ZORBAX SB-C18, 5um, 4.6x 250mm), mobile phase pH 1.5 perchloric acid: acetonitrile = 9:1, flow rate 0.5mL / min, UV detection wavelength 210nm.

[0065] The liquid phase spectra of substrate DL-p-hydroxyphenylglycine (DL-HPH), intermediate N-carbamoyl-D-p-hydroxyphenylglycine (CpHPG) and product D-p-hydroxyphenylglycine (D-HPG) are shown in Figure 2. Figure 4 shown.

[0066] The whole cells (20 g / L wet cells) of the obtained E. coli 01 were used to prepare D-p-hydroxyphenylglycine. The results were as follows: the yield of 30 g / L D-p-hydroxyphenylglycine after 12 hours of conversion reached 24.9 g / L, with a molar conversion rate of 95.4%, and the yield after 24 hours of conversion reached 25.4 g / L, with a molar conversion rate of 97.3%; the yield of 40 g / L D-p-hydroxyphenylglycine after 12 hours of conversion reached 30.4 g / L, with a molar conversion rate of 87.4%, and the yield after 24 hours of conversion reached 30.8 g / L, with a molar conversion rate of 88.5%; the yield of 50 g / L D-p-hydroxyphenylglycine after 12 hours of conversion reached 29.8 g / L, with a molar conversion rate of 68.5%, and the yield after 24 hours of conversion reached 33.0 g / L, with a molar conversion rate of 75.9%. The yield of D-p-hydroxyphenylglycine of 30 g / L substrate at different time periods is shown in Figure 2. Figure 5 ; The yield of D-p-hydroxyphenylglycine generated by conversion for 12h and 24h at substrate concentrations of 30g / L, 40g / L and 50g / L is shown in Figure 6 .

[0067] Embodiment 5:

[0068] Before obtaining E.coli 01, the present invention has designed a variety of expression methods:

[0069] Eight plasmids were constructed according to the similar method of Example 1: pACYCduet-1-GsDH-AkDC, pCDFduet-1-GsDH-AkDC, pETduet-1-GsDH-AkDC, pRSFduet-1-GsDH-AkDC, pACYCduet-1-AkDC–GsDH, pCDFduet-1-AkDC–GsDH, pETduet-1-AkDC–GsDH, pRSFduet-1-AkDC–GsDH, and then introduced into Escherichia coli BL21 (DE3) respectively, and the methods of Examples 2 and 3 were carried out, and the protein expression effect was observed by SDS-PAGE experiment. Their transformation effects were investigated in the manner of Example 4.

[0070] The expression results are as follows Figure 2 , conversion effect such as Figure 3 As shown by Figure 2 It can be seen that among the different double-fragment expression plasmids, when AkDC is placed after the first promoter, its supernatant expression level is significantly increased. Figure 3 It can be seen that in the 20 g / L reaction, pETduet-1 had the best effect, so the pETduet-1 vector was used.

[0071] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A D-p-hydroxyphenylglycine high-yielding strain, characterized in that: The strain uses plasmid pETduet-1 as a vector to express D-hydantoinase and D-carbamoylase mutant in an Escherichia coli host. The amino acid sequence of the D-hydantoinase is shown in SEQ ID NO.1, and the amino acid sequence of the D-carbamoylase mutant is shown in SEQ ID NO.

5.

2. The D-p-hydroxyphenylglycine high-yielding strain according to claim 1, characterized in that: The E. coli host is E. coli E. coli BL21 (DE3).

3. A method for constructing a high-yield D-p-hydroxyphenylglycine strain according to claim 1 or 2, characterized in that: The steps include: (1) Obtaining gene fragments encoding D-carbamoylase mutants and D-hydantoinase; (2) Connect the gene fragment encoding the D-carbamoylase mutant to the pETduet-1 vector, wherein the D-carbamoylase mutant gene fragment is located between the restriction sites BamHI and EcoRI. The resulting recombinant plasmid is pETduet-1- AkDC A273T ; (3) Introducing the recombinant plasmid described in step (2) into Escherichia coli E. coli BL21 (DE3), culture and extract plasmids to verify and enrich the recombinant plasmid pETduet-1- AkDC A273T ; (4) Connect the gene fragment encoding D-hydantoinase to the recombinant plasmid pETduet-1- AkDC A273T In which, the gene fragment of D-hydantoinase is located at the restriction site XhoⅠ, and the resulting recombinant plasmid is pETduet-1- AkDC A273T - GDJ ; (5) Transform the recombinant plasmid obtained in step (4) into E. coli BL21 (DE3), the D-p-hydroxyphenylglycine high-producing strain is obtained.

4. Use of the D-p-hydroxyphenylglycine high-yielding strain according to claim 1 or 2 in producing D-p-hydroxyphenylglycine.

5. The use according to claim 4, characterized in that: The application is to use the whole cells of the D-p-hydroxyphenylglycine high-yield strain after fermentation as a catalyst to catalyze DL-p-hydroxyphenylglycine to produce D-p-hydroxyphenylglycine.

6. The use according to claim 5, characterized in that: The fermentation of D-p-hydroxyphenylglycine high-yielding strain is carried out by inoculating the D-p-hydroxyphenylglycine high-yielding strain into a seed liquid culture medium to obtain a seed liquid, and then inoculating the seed liquid into a fermentation medium for cultivation.

7. The use according to claim 6, characterized in that: The fermentation culture conditions are: temperature 35~38 ℃, rotation speed 200~250 rpm, culture for 2~3 h, adding 0.3~0.5 mmol / L IPTG for induction, induction temperature 22~26 ℃, time 10~20 h, rotation speed 200~250 rpm.

8. The use according to claim 5, characterized in that: The conditions for the catalytic reaction are 38-42°C, 180-250 rpm, and a reaction pH of 7.5-8.0.

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