An engineered strain of Arthrobacter simplex with strong substrate transport and C1,2 response capabilities

Abstract

The present invention relates to an engineered strain of simple arthrobacter with strong transport ability and stress tolerance. The transport ability and stress tolerance of the strain are improved by overexpressing a transport protein in simple arthrobacter, or coexpressing a transport protein and a heat shock protein. The obtained engineered strain still maintains good catalytic activity in a conversion system of a high-concentration substrate and an organic solvent, and the C1,2 dehydrogenation reaction yield is increased by 1.42%-51.74% compared with a control strain. This engineered strain with strong transport ability and stress tolerance increases the concentration of substrates and organic solvents in the conversion system, thereby increasing the amount of product generated, and has important guiding significance for improving the conversion efficiency of high-concentration substrates of steroidal hydrophobic compounds.

Description

[0001] This application is patent number 202110228252X, the application date is 2021 / 3 / 2, and the invention name is: Division of an engineered strain of simple Arthrobacter with strong transport ability and stress tolerance. Technical field:

[0002] The invention belongs to the technical field of genetic engineering and microbial transformation, and specifically relates to a construction method of a simple Arthrobacter engineering strain with strong transport ability and stress tolerance, the strain and application thereof. Background technology:

[0003] Steroid hormone drugs are the second largest category of drugs in clinical use after antibiotics. They have anti-inflammatory, anti-allergic, anti-shock, and anti-allergic effects. In addition, steroid hormones also have the effects of improving protein metabolism, restoring and enhancing physical strength, and acting as diuretics and lowering blood pressure. The C1,2 dehydrogenation reaction of steroid compounds is a typical representative of the industrial production of steroid drugs using microbial transformation methods, and is also the most valuable reaction for the production of prednisolone and its homologues. Arthrobacter simplex is a steroid C1,2 dehydrogenation reaction strain commonly used in domestic industry. It has the advantages of high specificity and fast reaction rate. For example, it shows high activity against cortisone acetate (CA), and the anti-inflammatory activity of the resulting product, prednisolone acetate (PA), is increased by 3-4 times. However, steroid compounds have poor water solubility (solubility is generally 10 -5 -10 -6 mol / L) and limited substrate transport capacity limit the effective contact between substrate and intracellular catalytic enzyme, thus restricting production efficiency. So far, adding organic solvents is a method commonly used to improve substrate solubility in industrial steroid biosynthesis processes. However, the amount of organic solvents used is strictly controlled because of their adverse effects on microorganisms, which greatly limits the amount of substrate in the conversion system and ultimately affects the yield. In addition, high concentrations of substrates and products in the conversion system can also inhibit the vitality of the strain, thereby affecting production efficiency.

[0004] The ABC superfamily transport system exists in many eukaryotic and prokaryotic organisms, participates in various physiological activities of organisms, and completes the transmembrane transport of various molecules in the form of active transport. Its transported substrates include sugars, amino acids, peptides, proteins, metal ions, drug molecules, etc. The MFS superfamily transporter protein is one of the largest membrane transporter protein families known so far. This family of membrane transporters is widely present in the entire biological world, and its functions are closely related to many life activities. As a membrane transporter protein, its basic function is to assist in the transmembrane transport of substances. Its substrates include monosaccharides, polysaccharides, amino acids, peptides, vitamins, enzyme cofactors, drug molecules, chromophores, bases and many other small molecules.

[0005] Heat shock proteins (HSPs) are a general stress response of cells to external stress. Under normal conditions, HSPs are mainly responsible for the folding of new proteins, assisting protein transmembrane transport, assembling or disassembling oligomeric protein structures, and promoting the degradation of unstable proteins to prevent protein aggregation. Under stress conditions, their role is to prevent the aggregation of damaged proteins and help them refold.

[0006] There are currently no reports on overexpression of a single transporter or coexpression of transporters and heat shock proteins in steroid C1,2 dehydrogenase strains. Summary of the invention:

[0007] The purpose of the present invention is to provide a method for constructing a simple Arthrobacter engineered strain with strong transport capacity and stress tolerance, the strain and its application, so as to solve the problems of low substrate transport capacity and poor environmental stress tolerance of existing steroid C1,2 dehydrogenation reaction strains.

[0008] The technical scheme of the present invention to achieve the above-mentioned purpose is as follows:

[0009] One of the technical solutions of the present invention is to provide a genetically engineered bacterium of simplex with strong transport ability, characterized in that: the genetically engineered bacterium is obtained by using simplex bacterium (Arthrobacter simplex) CPCC140451 as a host cell and overexpressing any gene shown in SEQ ID No.3 in the sequence table.

[0010] Moreover, the transport protein corresponding to SEQ ID No.9 is ABC, and the nucleotide sequence of its encoding gene abc is shown in SEQ ID No.1; the transport protein corresponding to SEQ ID No.10 is MFS, and the nucleotide sequence of its encoding gene mfs is shown in SEQ ID No.2; the transport protein corresponding to SEQ ID No.11 is MB1, and the nucleotide sequence of its encoding gene mb1 is shown in SEQ ID No.3; the transport protein corresponding to SEQ ID No.12 is MB2, and the nucleotide sequence of its encoding gene mb2 is shown in SEQ ID No.4; the transport protein corresponding to SEQ ID No.13 is AMceG, and the nucleotide sequence of its encoding gene AmceG is shown in SEQ ID No.5; the transport protein corresponding to SEQ ID No.14 is AYrbE4A, and the nucleotide sequence of its encoding gene AyrbE4A is shown in SEQ ID No.6; the transport protein corresponding to SEQ ID No.15 is AYrbE4B, and the nucleotide sequence of its encoding gene AyrbE4B is shown in SEQ ID No.7; the genetically engineered bacteria uses pART2 plasmid as an expression vector;

[0011] The second technical solution of the present invention is to provide a genetically engineered strain of simple Arthrobacter with strong transport capacity and stress tolerance, which co-expresses a transporter gene and a heat shock protein encoding gene in simple Arthrobacter; the transporter gene is any one of the genes shown in No. 1-7. The heat shock protein encoding gene is groEL, which is the nucleotide sequence shown in SEQ ID No. 8, encoding a heat shock protein (GroEL), and the size of GroEL is 543 amino acids, and the sequence is shown in SEQ ID No. 16.

[0012] The genetically engineered bacteria uses the pART2 plasmid as an expression vector.

[0013] The third technical solution provided by the present invention is a method for constructing the above-mentioned genetically engineered bacteria with strong transport ability, which is specifically as follows:

[0014] (1) Using the genome of Arthrobacter simplex CPCC 140451 as a template, the transporter encoding genes abc, mfs, mb1, mb2, AmceG, AyrbE4A, and AyrbE4B in the strain were amplified by PCR;

[0015] (2) using genetic engineering methods to connect the above transporter protein encoding genes to the promoter of the Escherichia coli-Arthrobacter shuttle plasmid pART2, and then transform them into Escherichia coli DH5α for replication;

[0016] (3) The recombinant plasmids were extracted and electroporated into Arthrobacter simplex CPCC 140451 for overexpression.

[0017] The fourth technical solution provided by the present invention is a method for constructing the above-mentioned genetically engineered bacteria with strong transport ability and stress tolerance, which is specifically as follows:

[0018] (1) Using the genome of Arthrobacter simplex CPCC 140451 as a template, the transporter encoding genes abc, mfs, mb1, mb2, AmceG, AyrbE4A, AyrbE4B and the heat shock protein encoding gene groEL in the strain were amplified by PCR;

[0019] (2) using genetic engineering methods to connect the genes encoding the transporter protein and heat shock protein in series to the promoter of the Escherichia coli-Arthrobacter shuttle plasmid pART2, and then transfer them into Escherichia coli DH5α for replication;

[0020] (3) The recombinant plasmids were extracted and electroporated into Arthrobacter simplex CPCC 140451 for overexpression.

[0021] When the genetically engineered bacteria are used for steroid C1,2 dehydrogenation reaction, the concentration of substrate cortisone acetate in the conversion system is 0-120 g / L, preferably 6-90 g / L, more preferably 10-30 g / L, and the concentration of ethanol is 4-15%, preferably 6-10%.

[0022] The beneficial results of the present invention are as follows:

[0023] The present invention constructs simple Arthrobacter engineering strains with strong transport capacity and stress tolerance by overexpressing transport proteins and coexpressing transport proteins and heat shock proteins. These strains still maintain good catalytic activity in the conversion system of high-concentration substrates and organic solvents and substrates, and their C1,2 dehydrogenation reaction yields are increased by 1.42%-51.74% compared with the control strains. Description of the drawings:

[0024] Figure 1 The transcription levels of abc, mfs, mb1, mb2, AmceG, AyrbE4A and AyrbE4B genes induced by the substrate cortisone acetate (CA);

[0025] Figure 2 PCR and restriction enzyme electrophoresis of the engineered strains of Arthrobacter simplex overexpressing abc, mfs, mb1, mb2, AmceG, AyrbE4A, and AyrbE4B;

[0026] Wherein, M: DL5000 marker, (a) is the PCR result of recombinant pART2-abc plasmid; (b) is the result of double digestion of recombinant pART2-abc plasmid by Kpn I / Xba I; (c) is the PCR result of recombinant pART2-mfs plasmid; (d) is the result of double digestion of recombinant pART2-mfs plasmid by BamH I / Xba I; (e) is the PCR result of recombinant pART2-mb1 plasmid; (f) is the result of double digestion of recombinant pART2-mb1 plasmid by Kpn I / Xba I; (g) is the result of PCR of recombinant pART2-mb2 plasmid; (h) is the result of double digestion of recombinant pART2-mb2 plasmid by Kpn I / Xba I; (i) is the result of PCR of recombinant pART2-AmceG plasmid; (j) is the result of PCR of recombinant pART2-AyrbE4A plasmid; (k) is the result of PCR of recombinant pART2-AyrbE4B plasmid;

[0027] Figure 3 The process curve of the control strain pART2 and the Arthrobacter simplex engineered strains overexpressing abc, mfs, mb1, and mb2 in converting cortisone acetate (CA) to prednisolone acetate (PA) in a 6 g / L substrate CA, 0% ethanol solubilization system;

[0028] Figure 4 The process curve of the control strain pART2 and the Arthrobacter simplex engineered strains overexpressing abc, mfs, mb1, and mb2 in converting cortisone acetate (CA) to prednisolone acetate (PA) in a 6 g / L substrate CA and 8% ethanol solubilization system;

[0029] Figure 5 The process curve of the control strain pART2 and the Arthrobacter simplex engineered strains overexpressing abc, mfs, mb1, and mb2 in converting cortisone acetate (CA) to prednisolone acetate (PA) in a 15 g / L substrate CA and 8% ethanol solubilization system;

[0030] Figure 6 The process curve of the control strain pART2 and the Arthrobacter simplex engineered strains overexpressing abc, mfs, mb1, and mb2 in converting cortisone acetate (CA) to prednisolone acetate (PA) in a 90 g / L substrate CA and 8% ethanol solubilization system;

[0031] Figure 7 The process curve of the control strain pART2 and the AmceG, AyrbE4A, AyrbE4B overexpressing Arthrobacter simplex engineered strains converting cortisone acetate (CA) into prednisone acetate (PA) in a 15g / L substrate CA, 8% ethanol solubilization system;

[0032] Figure 8 PCR and restriction enzyme electrophoresis of the engineered strain of Arthrobacter simplex co-expressing abc and groEL genes;

[0033] Among them, (a) is the result of PCR of recombinant pART2-groEL-abc plasmid; (b) is the result of EcoR I / Xba I double enzyme digestion of recombinant pART2-groEL-abc plasmid;

[0034] Fig. 9 The process curve of the control strain pART2 and the groEL-abc co-expressing Arthrobacter simplex engineered strain converting cortisone acetate (CA) into prednisolone acetate (PA) in a 6g / L substrate CA, 8% ethanol solubilization system;

[0035] Fig.10 The process curve of the control strain pART2 and the groEL-abc co-expressing Arthrobacter simplex engineered strain converting cortisone acetate (CA) into prednisone acetate (PA) in a 15g / L substrate CA, 8% ethanol solubilization system;

[0036] Fig.11Process curve of the control strain pART2 and the groEL-abc co-expressing simplex bacterium engineered strain converting cortisone acetate (CA) into prednisone acetate (PA) in a 90g / L substrate CA and 8% ethanol solubilization system. Specific implementation method:

[0037] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0038] Example 1

[0039] Identification of proteins involved in substrate transport

[0040] The bacterial cells of Arthrobacter simplex CPCC 140451 were collected after being cultured for 3 hours with or without the addition of 6 g / L steroidal substrate CA, and the total RNA was extracted for transcriptomic sequencing. The results showed that compared with the absence of substrate addition, the addition of 6 g / L substrate CA led to a significant upregulation of the transcription levels of a series of genes, including: ABC transporter ATP binding protein encoding gene abc, MFS transporter encoding gene mfs, membrane protein encoding gene mb1, membrane protein encoding gene mb2, ABC transporter ATP binding protein encoding gene AmceG, ABC transporter permease encoding gene AyrbE4A, ABC transporter permease encoding gene AyrbE4B. Therefore, it is speculated that these proteins may be related to substrate transport. The transcription levels of the above seven transport-related protein encoding genes abc, mfs, mb1, mb2, AmceG, AyrbE4A, and AyrbE4B were further analyzed by qRT-PCR. Figure 1 It can be seen that after adding 6g / L substrate CA, the transcription levels of seven genes, abc, mfs, mb1, mb2, AmceG, AyrbE4A, and AyrbE4B, were upregulated by 3.95-336.79 times, indicating that these seven proteins are important proteins related to the substrate transport of simplex Arthrobacter.

[0041] Example 2

[0042] Construction of an engineered strain of Arthrobacter simplex overexpressing transporter-related genes

[0043] 2.1 Construction of recombinant plasmids pART2-abc, pART2-mfs, pART2-mb1, pART2-mb2, pART2-AmceG, pART2-AyrbE4A and pART2-AyrbE4B

[0044] Using G +Bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.) was used to extract the total DNA of Arthrobacter simplex CPCC 140451. The extracted total DNA was used as a template to amplify the abc, mfs, mb1, mb2, AmceG, AyrbE4A, and AyrbE4B genes. The primer sequences and the corresponding codes of the sequence table are shown in the following table:

[0045]

[0046] Note: The underline represents the restriction site

[0047] The PCR reaction system is shown in the following table (50 μL):

[0048]

[0049] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 2 min, 95°C for 20 s, 55-65°C for 20 s, 72°C for 2 min, and 72°C for 5 min after 35 cycles. After the PCR was completed, the products were tested by agarose gel electrophoresis to determine whether the fragment size was consistent.

[0050] The PCR product was purified using a DNA purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), and then digested with enzymes. The reaction system is as follows:

[0051]

[0052]

[0053] Incubate at 37°C for 2 h.

[0054] The prepared target gene fragments were ligated with the enzyme-digested and purified vector plasmid pART2 (GenBanK accession number: DQ191047) in a ratio of 1:3 (molar ratio) (16°C, 12h). The ligation products were transformed into Escherichia coli DH5α, and the correct recombinant plasmids pART2-abc, pART2-mfs, pART2-mb1, pART2-mb2, pART2-AmceG, pART2-AyrbE4A and pART2-AyrbE4B were obtained through PCR, enzyme digestion and sequencing techniques.

[0055] 2.2 Construction of engineering strains Asp / pART2-abc, Asp / pART2-mfs, Asp / pART2-mb1, Asp / pART2-mb2, Asp / pART2-AmceG, Asp / pART2-AyrbE4A and Asp / pART2-AyrbE4B.

[0056] (1) Preparation of Arthrobacter simplex competent cells

[0057] Arthrobacter simplex CPCC 140451 was selected and inoculated into LB liquid medium and cultured at 32°C and 160 rpm for 30 h until the bacterial OD 600 2.5 to form seed solution, take 1 mL of seed solution and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium, and culture at 32°C and 160 r / min for 8-10 h until OD 600 The concentration of penicillin G (70 μg / mL) as a cell wall treatment agent was 0.8-1.0, and the mixture was shaken for 1 hour. The flask containing the bacterial solution was placed on an ice bath for 20 minutes, and centrifuged at 6500 r / min for 10 minutes at 4°C, and the supernatant was discarded. 40 mL of electroporation buffer (composed of 15% glycerol and 0.5 mol / L sorbitol) precooled to 0°C was added to wash the bacteria, and the cells were centrifuged at 5000 r / min for 10 minutes at 4°C, and the supernatant was discarded. After repeated washing twice, 1.2 mL of electroporation buffer was added to resuspend the bacteria, and the cells were shaken to obtain simple bacterium competent cells, which were stored at -80°C for use.

[0058] (2) Electroporation and verification of recombinant plasmids

[0059] Take 120μL of simple Arthrobacter competent cells in a 1.5mL centrifuge tube, add 200ng of the constructed recombinant plasmid, mix well and transfer to a pre-cooled electric pulse cup in an ice bath for 3min; turn on the electric pulse instrument and perform electric shock transformation at 2.1kV; immediately add 1.2mL of sterile recovery medium (LB liquid medium containing 0.5moL / L sorbitol) to the electric pulse cup after electric shock, mix well, slowly shake and culture at 32℃ for 11h, then spread on a selection plate containing 50μg / mL kanamycin, invert and culture at 32℃ for 72h, and pick the transformant. Extract the plasmid for PCR and double enzyme digestion verification, and the verification results are as follows Figure 2 As shown, it indicates that the engineered strain has successfully expressed the corresponding gene. The successfully verified Arthrobacter simplex engineered strains were sent to Jinweizhi Biotechnology Co., Ltd. for further sequencing verification, and seven Arthrobacter simplex engineered strains that overexpressed the transporter encoding genes abc, mfs, mb1, mb2, AmceG, AyrbE4A, and AyrbE4B were obtained, named Asp / pART2-abc, Asp / pART2-mfs, Asp / pART2-mb1, Asp / pART2-mb2, Asp / pART2-AmceG, Asp / pART2-AyrbE4A, and Asp / pART2-AyrbE4B, respectively.

[0060] Example 3

[0061] Analysis of steroid C1,2 dehydrogenation ability of control strain pART2 and abc, mfs, mb1, mb2 overexpression engineering strains

[0062] The abc, mfs, mb1, mb2 overexpression engineering strains and the control strain pART2 constructed in Example 2 were picked from the slant and inoculated into LB liquid medium containing 50 μg / mL kanamycin. The culture was shaken at 32°C and 160 r / min for 36 h. A certain inoculum amount was transferred to a 250 mL triangular flask containing 50 mL of the above fresh medium. The initial OD 600 The value was adjusted to 0.2, and the culture was shaken at 32°C and 160r / min until the logarithmic growth phase. The substrate cortisone acetate (CA) with a final concentration of 0.1g / L was added to induce the production of C1,2 dehydrogenase, and the culture was shaken at 32°C and 160r / min for 18h until the middle and late logarithmic phase of each strain. The culture solution was centrifuged at 4°C and 6000r / min for 10min, and the collected bacteria were washed twice with pre-cooled 0.1M KH2PO4-NaOH solution (PBS buffer solution) at pH 7.2, and the bacteria were suspended in an appropriate amount of PBS buffer to prepare resting cells.

[0063] Use the above resting cells to prepare 30 mL of transformation system:

[0064] Transformation system I: Bacteria OD 600 is 2.0, the substrate CA concentration is 6 g / L, and 0% ethanol is used as the solubilizer; the conversion process curve is shown in Figure 3 As shown;

[0065] Transformation system II: bacterial OD 600 is 2.0, the substrate CA concentration is 6 g / L, and 8% ethanol is used as the solubilizer; the conversion process curve is shown in Figure 4 As shown;

[0066] Transformation system III: bacterial OD 600 is 2.0, the substrate CA concentration is 15g / L, and 8% ethanol is used as the solubilizer; the conversion process curve is shown in Figure 5 As shown;

[0067] Transformation system IV: bacterial OD 600 is 2.0, the substrate CA concentration is 90g / L, and 8% ethanol is used as the solubilizer; the conversion process curve is shown in Figure 6 shown.

[0068] The reaction was carried out at 34°C and 180 r / min, and the concentration of the product prednisone acetate (PA) was determined by sampling at regular intervals. 0.4 mL of the sample was added to 0.8 mL of ethyl acetate to terminate the reaction, and ultrasonic extraction was performed for more than 10 min. The sample was centrifuged at 12000 r / min for 10 min, and 100 μL of the supernatant was taken into a new 1.5 ml centrifuge tube, evaporated overnight in a fume hood, and then re-dissolved with 1 mL of mobile phase. The conversion rate of the substrate CA and the amount of the product PA were determined by HPLC.

[0069] HPLC detection conditions are:

[0070] High performance liquid chromatograph: Agilent 1100 Series LC (G1314 Pump, G1322A DEGASSER G1314 VWD detector, 10 μL A N injector, HP ChemStation);

[0071] Chromatographic column: Kromasil 100-5SIL 250 mm × 4.6 mm × 5 μm;

[0072] Mobile phase: dichloromethane: ether: methanol (volume ratio 86:12:2), filtered through a 0.45 μm microporous membrane;

[0073] Flow rate: 1 mL / min;

[0074] Column temperature: 30°C;

[0075] Detector: UVDetector, wavelength: 240nm.

[0076] Injection volume: 20 μL

[0077] Samples were taken at different times during the conversion process and the results are shown in Table 1.

[0078] Table 1 Comparison of the conversion results of cortisone acetate to prednisone acetate by the transporter overexpression engineered strain and the control strain

[0079]

[0080]

[0081] As shown in Table 1, in transformation system I, the concentration of PA generated by the abc and mb1 overexpressing engineering bacteria was higher than that of the control strain pART2, which increased by 10.26% and 4.14%, respectively; in transformation system II, the concentration of PA generated by the abc and mb1 overexpressing engineering bacteria was higher than that of the control strain pART2, which increased by 4.40% and 3.48%, respectively; in transformation system III, the concentration of PA generated by the abc, mfs, mb1, and mb2 overexpressing engineering bacteria was significantly higher than that of the control strain pART2, and the order from high to low was abc (19.70%) > mb1 (6.29%) > mb2 (5.58%) > mfs (1.42%); in transformation system III, the concentration of PA generated by the mfs, mb1, and mb2 overexpressing engineering bacteria was significantly higher than that of the control strain pART2, which increased by 21.79%, 51.74%, and 50.67%, respectively.

[0082] Example 4

[0083] Analysis of steroid C1,2 dehydrogenation ability of control strain pART2 and AmceG, AyrbE4A, and AyrbE4B overexpressing engineered strains

[0084] The AmceG, AyrbE4A, AyrbE4B overexpression engineering strains and the control strain pART2 constructed in Example 2 were picked from the slant and inoculated into LB liquid medium containing 50 μg / mL kanamycin. The culture was shaken at 32°C and 160 r / min for 36 h. A certain inoculum amount was transferred to a 250 mL Erlenmeyer flask containing 50 mL of the above fresh medium. The initial OD 600 The value was adjusted to 0.2, and the culture was shaken at 32°C and 160r / min until the logarithmic growth phase. The substrate cortisone acetate (CA) with a final concentration of 0.1g / L was added to induce the production of C1,2 dehydrogenase, and the culture was shaken at 32°C and 160r / min for 18h until the middle and late logarithmic phase of each strain. The culture solution was centrifuged at 4°C and 6000r / min for 10min, and the collected bacteria were washed twice with pre-cooled 0.1M KH2PO4-NaOH solution (PBS buffer solution) at pH 7.2, and the bacteria were suspended in an appropriate amount of PBS buffer to prepare resting cells.

[0085] Prepare 30 mL of transformation system using the above resting cells: bacterial OD 600The concentration of substrate CA was 2.0, the concentration of substrate CA was 15g / L, and 8% ethanol was used as the solubilizer; the reaction was oscillated at 34°C and 180r / min, and the concentration of product prednisone acetate (PA) was determined by sampling at regular intervals. Each time 0.4mL of the sample was added with 0.8mL of ethyl acetate to terminate the reaction, ultrasonic extraction was performed for more than 10min, and the sample was centrifuged at 12000r / min for 10min. 100μL of the supernatant was taken into a new 1.5ml centrifuge tube, evaporated overnight in a fume hood, and then re-dissolved with 1mL of mobile phase. The conversion rate of substrate CA and the amount of product PA generated were determined by HPLC.

[0086] The HPLC detection conditions are the same as those in Example 3.

[0087] Samples were taken at different times during the transformation process, and the transformation process curve was as follows: Figure 7 The results are shown in Table 2.

[0088] Table 2 Comparison of the conversion results of cortisone acetate to prednisone acetate by the AMceG, AYrbE4A, and AYrbE4B overexpressing engineering strains and the control strains

[0089]

[0090] As can be seen from Table 2, in the conversion system with a substrate CA concentration of 15 g / L and an ethanol concentration of 8%, the concentrations of CA converted to PA by AmceG, AyrbE4A, and AyrbE4B overexpressing engineered bacteria were significantly higher than those of the control strain pART2, and the order from high to low was AmceG (13.52%) > AyrbE4A (8.53%) > AyrbE4B (4.26%).

[0091] Example 5

[0092] Construction of a genetically engineered strain of Arthrobacter simplex co-expressing transporter and heat shock protein genes

[0093] 5.1 Construction of recombinant plasmid pART2-groEL

[0094] Using G + Bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.) was used to extract the total DNA of Arthrobacter simplex CPCC 140451. The extracted total DNA was used as a template to amplify the groEL gene. The primer sequences and the corresponding codes of the sequence table are shown in the following table:

[0095]

[0096]

[0097] Note: The underline represents the restriction site

[0098] The PCR reaction system is shown in the following table (50 μL):

[0099]

[0100] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 2 min, 95°C for 20 s, 55-65°C for 20 s, 72°C for 2 min, and 72°C for 5 min after 35 cycles. After the PCR was completed, the products were tested by agarose gel electrophoresis to determine whether the fragment size was consistent.

[0101] The PCR product was purified using a DNA purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), and then digested with enzymes. The reaction system is as follows:

[0102]

[0103] Incubate at 37°C for 2 h.

[0104] The prepared target fragments were ligated with the vector plasmid pART2 after enzyme digestion and purification at a ratio of 1:3 (16°C, 12h), and the ligation products were transformed into Escherichia coli DH5α, and the recombinant plasmid pART2-groEL was obtained after screening.

[0105] 5.2 Construction of recombinant plasmid pART2-groEL-abc

[0106] The promoter and abc gene were amplified using the recombinant plasmid pART2-abc as a template. The primer sequences and the corresponding codes in the sequence table are shown in the following table:

[0107]

[0108] The PCR reaction system is shown in the following table (50 μL):

[0109]

[0110] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 2 min, 95°C for 20 s, 55°C for 20 s, 72°C for 2 min, and 72°C for 5 min after 35 cycles. After the PCR was completed, the products were tested by agarose gel electrophoresis to determine whether the fragment size was consistent.

[0111] The PCR product was purified using a DNA purification kit (Shanghai Shenggong Biotechnology Service Co., Ltd.). The recombinant plasmid pART2-groEL was digested with enzymes, and the reaction system was as follows:

[0112]

[0113] Incubate at 37°C for 2 h.

[0114] The prepared target fragments were respectively connected with the vector plasmid pART2-groEL after enzyme digestion and purification using the Gibson assembly method (55°C, 15 min), and the connection products were transformed into Escherichia coli DH5α, and the recombinant plasmid pART2-groEL-abc was obtained after screening.

[0115] 5.3 Construction of the engineered strain Asp / pART2-groEL-abc co-expressing Arthrobacter simplex

[0116] (1) Preparation of Arthrobacter simplex competent cells

[0117] The method for preparing competent cells of Arthrobacter simplex is as described in Example 2.

[0118] (2) Electroporation and verification of recombinant plasmids

[0119] Take 120μL Arthrobacter simplex competent cells in a 1.5mL centrifuge tube, add 200ng of the constructed recombinant plasmid pART2-groEL-abc, mix well, transfer to a pre-cooled electric pulse cup and place in an ice bath for 3min; turn on the electric pulse instrument and perform electric shock transformation at 2.1kV; immediately add 0.8mL of sterile recovery medium (LB liquid medium containing 0.5moL / L sorbitol) to the electric pulse cup after electric shock, mix well, slowly shake and culture at 32℃ for 11h, then spread on a selection plate containing 50μg / mL kanamycin, invert and culture at 32℃ for 72h, and pick the transformant. Extract the plasmid for PCR and double enzyme digestion verification, and then Figure 8 It can be seen that PCR amplification has a band around 2600bp, which is consistent with the length of the target fragment "groEL (1632bp) + promoter (218bp) + abc (759bp)" 2609bp; the plasmid has bands around 4000bp and 3200bp after EcoR I and Xba I double digestion, which are consistent with the vector (3994bp) and fragment (3205bp) size, respectively, indicating that the engineering strain has successfully expressed the groEL and abc genes. It was sent to Jinweizhi Biotechnology Co., Ltd. for further sequencing and verification, and the verified engineering strain was named Asp / pART2-groEL-abc.

[0120] Example 6

[0121] Analysis of the Steroid C1,2 Dehydrogenation Reaction Ability of the Engineered Strains of Arthrobacter simplex Co-expressing with the Control Strain pART2 and groEL-abc

[0122] The groEL-abc co-expression engineering strain constructed in Example 5 and the control strain pART2 were picked from the slant and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 32°C, 160 r / min, and shaken for 36 h. A certain inoculum amount was transferred to a 250 mL triangular flask containing 50 mL of the above fresh medium. The initial OD 600 The value was adjusted to 0.2, and the culture was shaken at 32°C and 160r / min until the logarithmic growth phase. The substrate cortisone acetate (CA) with a final concentration of 0.1g / L was added to induce the production of C1,2 dehydrogenase, and the culture was shaken at 32°C and 160r / min for 18h until the middle and late logarithmic phase of each strain. The culture solution was centrifuged at 4°C and 6000r / min for 10min, and the collected bacteria were washed twice with pre-cooled 0.1M KH2PO4-NaOH solution (PBS buffer solution) at pH 7.2, and the bacteria were suspended in an appropriate amount of PBS buffer to prepare resting cells.

[0123] Use the above resting cells to prepare 30 mL of transformation system:

[0124] Transformation system I: Bacteria OD 600 is 2.0, the substrate CA concentration is 6 g / L, and 8% ethanol is used as the solubilizer; the conversion process curve is shown in Fig. 9 As shown;

[0125] Transformation system II: bacterial OD 600 is 2.0, the substrate CA concentration is 15g / L, and 8% ethanol is used as the solubilizer; the conversion process curve is shown in Fig.10 As shown;

[0126] Transformation system III: bacterial OD 600 is 2.0, the substrate CA concentration is 90g / L, and 8% ethanol is used as the solubilizer; the conversion process curve is shown in Fig.11 shown.

[0127] The reaction was carried out at 34°C and 180 r / min, and the concentration of the product prednisone acetate (PA) was determined by sampling at regular intervals. 0.4 mL of the sample was added to 0.8 mL of ethyl acetate to terminate the reaction, and ultrasonic extraction was performed for more than 10 min. The sample was centrifuged at 12000 r / min for 10 min, and 100 μL of the supernatant was taken into a new 1.5 ml centrifuge tube, evaporated overnight in a fume hood, and then re-dissolved with 1 mL of mobile phase. The conversion rate of the substrate CA and the amount of the product PA were determined by HPLC.

[0128] The HPLC detection conditions are the same as those in Example 3.

[0129] Samples were taken at different times during the conversion process and the results are shown in Table 3.

[0130] Table 3 Comparison of the transformation results of cortisone acetate to prednisone acetate by groEL-abc co-expression engineered strain and control strain

[0131]

[0132] As shown in Table 3, in transformation system I, the PA production of abc and groEL co-expressing engineering bacteria reached a maximum of 5.58 g / L at 6 h of transformation, which was 2.20% higher than the control strain (5.46 g / L); in transformation system II, the PA production of abc and groEL co-expressing engineering bacteria reached a maximum of 12.58 g / L at 36 h of transformation, which was 27.72% higher than the control strain (9.85 g / L), which was higher than the abc overexpressing engineering bacteria (18.27%) in transformation system III of Example 3, and the transformation time was shortened from 48 h to 36 h; in transformation system III, the PA production of abc and groEL co-expressing engineering bacteria reached a maximum of 22.19 g / L at 108 h of transformation, which was 19.11% higher than the control strain (18.63 g / L).

[0133] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the attached claims.

Claims

1. An engineered strain of Arthrobacter simplex with strong substrate transport and C1,2 reaction ability, characterized by: Arthrobacter simplex ( Arthrobacter simplex ) CPCC 140451 is a host cell, which is obtained by overexpressing the gene shown in SEQ ID No. 3 in the sequence table.

2. The simple Arthrobacter engineered strain with strong substrate transport and C1,2 reaction ability according to claim 1, characterized in that: The genetically engineered bacteria uses the pART2 plasmid as an expression vector.

3. A method for constructing an engineered strain of Arthrobacter simplex with strong substrate transport and C1,2 reaction ability as claimed in claim 1, characterized in that: The steps are as follows: (1) Using the genome of Arthrobacter simplex CPCC 140451 as a template, the transporter protein encoding gene in the strain was amplified by PCR, and the gene was the gene shown in SEQ ID No. 3; (2) Using genetic engineering methods, the above transporter protein encoding genes were connected to the promoter of the Escherichia coli-Arthrobacter shuttle plasmid pART2, and then transferred to Escherichia coli DH5α for replication; ⑶ The extracted recombinant plasmids were electroporated into Arthrobacter simplex CPCC 140451 for overexpression.

4. A method for catalyzing the dehydrogenation reaction of cortisone acetate at the C1,2 position, characterized in that: The simplex Arthrobacter engineered strain with strong substrate transport and C1,2 reaction ability as claimed in any one of claims 1 to 3 is used to carry out the conversion of cortisone acetate substrate.

5. An engineered strain of Arthrobacter simplex with strong substrate transport and C1,2 reaction ability, characterized by: The engineered bacteria are simple Arthrobacter ( Arthrobacter simplex ) CPCC 140451 was used as the host cell, and the pART2 plasmid was used as the expression vector, and the gene shown in SEQ ID No. 3 and the heat shock protein encoding gene shown in SEQ ID No. 8 were co-expressed.

6. A method for constructing an engineered strain of Arthrobacter simplex with strong substrate transport and C1,2 reaction ability as claimed in claim 5, characterized in that: The steps are as follows: (1) Using the genome of Arthrobacter simplex CPCC 140451 as a template, the transporter encoding gene and the heat shock protein encoding gene in the strain were amplified by PCR, the transporter encoding gene was the gene shown in SEQ ID No.3, and the heat shock protein encoding gene was the gene shown in SEQ ID No.8; (2) Using genetic engineering methods, the coding genes of the above transporter protein and heat shock protein are connected in series to the promoter of the Escherichia coli-Arthrobacter shuttle plasmid pART2, and then transferred to Escherichia coli DH5α for replication; ⑶ The extracted recombinant plasmids were electroporated into Arthrobacter simplex CPCC 140451 for overexpression.

7. A method for catalyzing the dehydrogenation reaction of cortisone acetate at the C1,2 position, characterized in that: The Arthrobacter simplex genetically engineered bacteria as described in any one of claims 5-6 are used to carry out the transformation of cortisone acetate substrate.

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