CN116590206A - Steroid C1,2 dehydrogenation reaction engineering strain with excellent transport and reaction activity - Google Patents

Steroid C1,2 dehydrogenation reaction engineering strain with excellent transport and reaction activity Download PDF

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CN116590206A
CN116590206A CN202211205623.3A CN202211205623A CN116590206A CN 116590206 A CN116590206 A CN 116590206A CN 202211205623 A CN202211205623 A CN 202211205623A CN 116590206 A CN116590206 A CN 116590206A
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arthrobacter
strain
part2
transport
gene
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骆健美
王敏
申雁冰
贾红晨
朱文成
宋昭玉
艾露
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Tianjin University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/02—Dehydrogenating; Dehydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/06—Arthrobacter
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/584—Recycling of catalysts

Abstract

The application relates to a steroid C1,2 dehydrogenation reaction engineering strain with excellent transport and reaction activity, which improves the transport capacity and stress tolerance of the strain by over-expressing transport protein or co-expressing transport protein and heat shock protein in Arthrobacter simplex, wherein the obtained engineering strain still maintains good catalytic activity in a conversion system of high-concentration substrate and organic solvent, and the yield of the C1,2 dehydrogenation reaction is improved by 1.42-51.74% compared with that of a control strain. The engineering strain with strong transport capacity and stress tolerance improves the concentration of the substrate and the organic solvent in a conversion system, further improves the product yield, and has important guiding significance for improving the conversion efficiency of the steroid hydrophobic compound high-concentration substrate.

Description

Steroid C1,2 dehydrogenation reaction engineering strain with excellent transport and reaction activity
The application is patent number 202110228252X, the application date is 2021/3/2, and the application is named as follows: a scheme of simple Arthrobacter engineering strains with strong transport capacity and stress tolerance is provided.
Technical field:
the application belongs to the technical fields of genetic engineering and microbial transformation, and particularly relates to a construction method of a simple Arthrobacter engineering strain with strong transport capacity and stress tolerance, a strain and application thereof.
The background technology is as follows:
the steroid hormone medicine is the second most clinically used medicine which is only inferior to antibiotics at present, has the effects of resisting inflammation, allergy, shock, allergic reaction and the like, and in addition, the steroid hormone has the effects of improving protein metabolism, recovering and enhancing physical strength, promoting urination, reducing blood pressure and the like. The C1,2 dehydrogenation reaction of steroids is a typical representation of the industrial production of steroid drugs by microbial conversion, and is also the most valuable reaction for the production of prednisolone and its homologs. Arthrobacter simplicissimus (Arthrobacter simplex) is a steroid C1,2 dehydrogenation reaction strain commonly used in domestic industry at present, and has the advantages of high specificity, high reaction rate and the like. If it shows high activity on Cortisone Acetate (CA), the anti-inflammatory activity of the product Prednisone Acetate (PA) is increased by 3-4 times. However, steroids have poor water solubility (solubility typically 10 -5 -10 -6 mol/L) and limited substrate transport capacity limit effective contact of the substrate with the intracellular catalytic enzyme, and thusThe production efficiency is restricted. The addition of organic solvents has so far been a method commonly used in the industrial steroid biosynthesis process to improve the solubility of the substrate. However, the amount of organic solvent used is severely controlled because of its adverse effect on the microorganism, which greatly limits the amount of substrate fed into the conversion system and ultimately affects the yield. In addition, high concentration of substrates and products in the transformation system can also inhibit the activity of the strain, thereby influencing the production efficiency.
The ABC superfamily transport system exists in many eukaryotic and prokaryotic organisms, and is involved in various physiological activities of the organisms, and performs transmembrane transport of various molecules in an active transport manner, and substrates for transport include sugar, amino acid, polypeptide, protein, metal ion, drug molecules and the like. MFS superfamily transporters are one of the largest families of membrane transporters currently known. The family of membrane transporters is widely found throughout the biological world, and their function is also closely related to many vital phenomena. As membrane transporters, the basic function is to assist in completing the transmembrane transport of substances, and substrates include monosaccharides, polysaccharides, amino acids, polypeptides, vitamins, enzyme cofactors, drug molecules, chromophores, bases and numerous small molecules.
Heat Shock Proteins (HSPs) are general stress reactions of cells to external pressure stress, and under normal conditions, HSPs are mainly responsible for folding of nascent proteins, assisting in transmembrane transport of proteins, assembling or splitting oligomeric protein structures, and promoting degradation of labile proteins to prevent protein aggregation. Under stress conditions, they function to prevent aggregation of damaged proteins and to assist their refolding.
There is no report on overexpression of single transporter, co-expression of transporter and heat shock proteins in steroid C1,2 dehydrogenation strains.
The application comprises the following steps:
the application aims to provide a construction method of a steroid C1,2 dehydrogenation reaction engineering strain with excellent transportation and reaction activity, the strain and application thereof. Thereby solving the problems of low substrate transfer capacity, poor environmental stress tolerance and the like of the existing steroid C1,2 dehydrogenation reaction strain.
The technical scheme for achieving the aim is as follows:
one of the technical schemes of the application is to provide a simple Arthrobacter genetic engineering bacterium with strong transfer capability, which is characterized in that: the genetically engineered bacterium is obtained by using Arthrobacter simplex (Arthrobacter simplex) CPCC 140451 as a host cell and over-expressing a gene shown in any one of SEQ ID No.1-7 of a sequence table.
In addition, the transporter corresponding to SEQ ID No.9 is ABC, and the nucleotide sequence of the coding gene ABC is shown as SEQ ID No. 1; the transport protein corresponding to SEQ ID No.10 is MFS, and the nucleotide sequence of the encoding gene MFS is shown as SEQ ID No. 2; the transport protein corresponding to SEQ ID No.11 is MB1, and the nucleotide sequence of the coding gene MB1 is shown as SEQ ID No. 3; the transport protein corresponding to SEQ ID No.12 is MB2, and the nucleotide sequence of the coding gene MB2 is shown as SEQ ID No. 4; the transport protein corresponding to SEQ ID No.13 is AMceG, and the nucleotide sequence of the coding gene AmceG is shown as SEQ ID No. 5; the transport protein corresponding to SEQ ID No.14 is AYrbE4A, and the nucleotide sequence of the encoding gene AYrbE4A is shown as SEQ ID No. 6; the transport protein corresponding to SEQ ID No.15 is AYrbE4B, and the nucleotide sequence of the encoding gene AYrbE4B is shown as SEQ ID No. 7; the genetic engineering bacteria take pART2 plasmid as an expression vector;
the second technical scheme of the application is to provide a simple arthrobacter genetic engineering strain with strong transport capacity and stress tolerance, which is to co-express a transporter gene and a coding gene of heat shock protein in simple arthrobacter; the transporter gene is a gene shown in any one of No. 1-7. The heat shock protein coding gene is groEL, the nucleotide sequence shown as SEQ ID No.8, the heat shock protein (GroEL) is coded, the GroEL size is 543 amino acids, and the sequence is shown as SEQ ID No. 16.
The genetic engineering bacteria take pART2 plasmid as an expression vector.
The third technical scheme provided by the application is a construction method of the genetically engineered bacterium with strong transport capacity, which comprises the following steps:
(1) The gene abc, mfs, mb, mb2, amceG and AyrbE4A, ayrbE B of the transporter coding genes in the strain are obtained by PCR amplification by taking the genome of Arthrobacter simplex CPCC 140451 as a template;
(2) The transporter coding genes are respectively connected to promoters of E.coli-arthrobacter shuttle plasmid pART2 by using a genetic engineering means, and then transformed into E.coli DH5 alpha for replication;
(3) The extracted recombinant plasmids are respectively and electrically transformed into Arthrobacter simplex CPCC 140451 for over-expression.
The fourth technical scheme provided by the application is a construction method of the genetically engineered bacterium with strong transport capacity and stress tolerance, which comprises the following steps:
(1) The gene abc, mfs, mb, mb2, amceG, ayrbE4A, ayrbE B and a heat shock protein coding gene groEL in the strain are obtained by PCR amplification by taking the genome of Arthrobacter simplex CPCC 140451 as a template;
(2) The coding genes of the transport protein and the heat shock protein are connected in series by utilizing a genetic engineering means and then are connected to a promoter of an escherichia coli-arthrobacter shuttle plasmid pART2, and then are transformed into escherichia coli DH5 alpha for replication;
(3) The extracted recombinant plasmids are respectively and electrically transformed into Arthrobacter simplex CPCC 140451 for over-expression.
When the genetically engineered bacterium is applied to the dehydrogenation reaction of steroid C1 and 2, the concentration of the substrate cortisone acetate in a conversion system is 0-120g/L, preferably 6-90g/L, and more preferably 10-30g/L. The ethanol concentration is 4-15%, preferably 6-10%.
The beneficial results of the application are as follows:
the application constructs the simple Arthrobacter engineering strain with strong transport capacity and stress tolerance through the over-expression of the transport protein and the co-expression of the transport protein and the heat shock protein. The strains still keep good catalytic activity in a conversion system of high-concentration substrates, organic solvents and substrates, and the yield of the C1 and 2 dehydrogenation reaction is improved by 1.42-51.74% compared with that of a control strain.
Description of the drawings:
FIG. 1 transcript levels of abc, mfs, mb1, mb2, amceG, ayrbE4A and AyrbE4B genes under the induction of the substrate Cortisone Acetate (CA);
FIG. 2 PCR and restriction map of engineering strains of Arthrobacter simplicissimi overexpressed by abc, mfs, mb1, mb2, amceG, ayrbE4A, ayrbE B;
wherein, M is DL5000 marker, (a) is PCR result of recombinant pART2-abc plasmid; (b) Is the result of double cleavage of recombinant pART2-abc plasmid by Kpn I/Xba I; (c) PCR results for recombinant pART2-mfs plasmid; (d) The result of BamH I/Xba I double digestion of the recombinant pART2-mfs plasmid; (e) PCR results for recombinant pART2-mb1 plasmid; (f) Is the result of double cleavage of recombinant pART2-mb1 plasmid by Kpn I/Xba I; (g) results of PCR for recombinant pART2-mb2 plasmid; (h) Is the result of double cleavage of recombinant pART2-mb2 plasmid by Kpn I/Xba I; (i) results of PCR for recombinant pART2-AmceG plasmid; (j) results of PCR for recombinant pART2-AyrbE4A plasmid; (k) results of PCR for recombinant pART2-AyrbE4B plasmid;
FIG. 3 Process curves for the conversion of Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 6g/L substrate CA,0% ethanol co-solution system by control strains pART2 and abc, mfs, mb1, mb2 over-expressing simple arthrobacter engineering strains;
FIG. 4 is a graph showing the process of converting Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 6g/L substrate CA,8% ethanol co-solution system by control strains pART2 and abc, mfs, mb1, mb2 over-expressing an engineering strain of Arthrobacter simplex;
FIG. 5 Process curves for the conversion of Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 15g/L substrate CA,8% ethanol co-solution system by control strains pART2 and abc, mfs, mb1, mb2 over-expressing simple arthrobacter engineering strains;
FIG. 6 Process curves for the conversion of Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 90g/L substrate CA,8% ethanol co-solution system by control strains pART2 and abc, mfs, mb1, mb2 over-expressing simple arthrobacter engineering strains;
FIG. 7A process curve for the conversion of Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 15g/L substrate CA,8% ethanol co-solution system by control strains pART2 and AmceG, ayrbE4A, ayrbE B over-expressing an engineering strain of Arthrobacter simplex;
FIG. 8 PCR and restriction map of a simple Arthrobacter engineering strain co-expressing abc and groEL genes;
wherein, (a) is the result of PCR on recombinant pART2-groEL-abc plasmid; (b) The result of EcoR I/Xba I double digestion of the recombinant pART2-groEL-abc plasmid;
FIG. 9A process curve for the conversion of Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 6g/L substrate CA,8% ethanol co-solution system by control strains pART2 and groEL-abc co-expressing Arthrobacter simplex engineering strains;
FIG. 10A graph of the process of converting Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 15g/L substrate CA,8% ethanol co-solution system by co-expressing Arthrobacter simplex engineering strains pART2 and groEL-abc;
FIG. 11A process curve for the conversion of Cortisone Acetate (CA) to Prednisone Acetate (PA) in a 90g/L substrate CA,8% ethanol co-solution system by control strains pART2 and groEL-abc co-expressing Arthrobacter simplex engineering strains.
The specific embodiment is as follows:
in order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present application.
Example 1
Determination of substrate transport-related proteins
The Arthrobacter simplex CPCC 140451 cells were cultured for 3 hours with and without 6g/L of steroid substrate CA, and total RNA was extracted for transcriptomic sequencing. The results indicate that the addition of 6g/L substrate CA resulted in a significant up-regulation of transcription levels of a range 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, and therefore, these proteins are presumed to be involved in substrate transport. The transcript levels of the seven transport-related protein encoding genes abc, mfs, mb1, mb2, ameg, ayrbE4A, ayrbE B were further analyzed using qRT-PCR. As can be seen from FIG. 1, the transcription levels of seven genes abc, mfs, mb, mb2, amceG, ayrbE4A, ayrbE B were up-regulated 3.95-336.79 fold after addition of 6g/L of substrate CA. These seven proteins are shown to be important proteins associated with simple arthrobacter substrate transport.
Example 2
Construction of transporter related gene over-expression simple arthrobacter engineering strain
2.1 construction of recombinant plasmids pART2-abc, pART2-mfs, pART2-mb1, pART2-mb2, pART2-AmceG, pART2-AyrbE4A and pART2-AyrbE4B
Using G + Bacterial genomic DNA extraction kit (Tiangen Biochemical technology Co., ltd.) total DNA of Arthrobacter simple CPCC 140451 was extracted. The abc, mfs, mb, mb2, amceG, ayrbE4A, ayrbE B genes were amplified using the extracted total DNA as templates, respectively. The primer sequences and the corresponding codes of the sequence table are shown in the following table:
note that: underline indicates the cleavage site
The PCR reaction system is shown in the following table (50. Mu.L):
the PCR reaction conditions were: the product was subjected to agarose gel electrophoresis to determine whether the fragment size was consistent after the PCR was completed, at 95℃for 2min,95℃for 20s,55-65℃for 20s,72℃for 2min, and 35 cycles for 72℃for 5 min.
The PCR product was purified using a DNA purification kit (Shanghai Biotechnology service Co., ltd.) and then digested, and the reaction system was as follows:
the reaction was carried out in a bath at 37℃for 2h.
The prepared target gene fragments are respectively subjected to ligation reaction (16 ℃ C., 12 h) with enzyme-digested and purified vector plasmid pART2 (GenBank accession number: DQ 191047) according to the ratio of 1:3 (the mass ratio), the ligation products are respectively transformed into escherichia coli DH5 alpha, and correct recombinant plasmids pART2-abc, pART2-mfs, pART2-mb1, pART2-mb2, pART2-AmceG, pART2-Ayrb 4A and pART2-Ayrb 4B are obtained through PCR, enzyme digestion and sequencing technologies.
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.
(1) Preparation of Arthrobacter simple competent cells
Inoculating Arthrobacter simplex CPCC 140451 into LB liquid medium, shake culturing at 32deg.C and 160r/min for 30 hr to obtain thallus OD 600 Forming seed solution for 2.5, taking 1mL of seed solution, inoculating into 250mL triangular flask containing 50mL LB liquid medium, culturing at 32deg.C and 160r/min for 8-10h, and reaching OD 600 Adding cell wall treating agent penicillin G (concentration of 70 μg/mL) into the solution for shaking treatment for 1h, cooling the triangular flask containing the bacterial liquid on ice bath for 20min, centrifuging for 10min at 6500r/min at 4deg.C, and discarding supernatant; after washing the cells with 40mL of shock buffer (composed of 15% glycerol and 0.5mol/L sorbitol) pre-cooled to 0deg.C, centrifuging at 4deg.C for 10min at 5000r/min, and discarding the supernatant; after repeated washing twice, 1.2mL of electric shock buffer solution is added to resuspend the thalli, and the thalli are uniformly shaken to obtain competent cells of the Arthrobacter simplex, and the competent cells are preserved at the temperature of minus 80 ℃ for standby.
(2) Recombinant plasmid electric shock transformation and verification
Taking 120 mu L of simple Arthrobacter competent cells in a 1.5mL centrifuge tube, respectively adding 200ng of constructed recombinant plasmid, uniformly mixing, and transferring to a precooled electric pulse cup for ice bath for 3min; turning on an electric pulse instrument, and converting 2.1kV electric shock; immediately after electric excitation, 1.2mL of sterile resuscitating medium (LB liquid medium containing 0.5moL/L sorbitol) was added into the electric pulse cup, after mixing, the mixture was slowly cultured by shaking at 32℃for 11 hours, and then the mixture was spread on a selection plate containing 50. Mu.g/mL kanamycin, and the mixture was cultured upside down at 32℃for 72 hours to pick up transformants. The plasmid was extracted for PCR and double enzyme digestion verification, and the verification result is shown in FIG. 2, which shows that the engineering strain has successfully expressed the corresponding gene. The simple Arthrobacter engineering strains which are successfully verified are all sent to Jinweizhi biotechnology limited company for further sequencing verification, and seven simple Arthrobacter engineering strains which are respectively over-expressed with transporter coding genes abc, mfs, mb, mb2, amceG and AyrbE4A, ayrbE B are obtained and are respectively named Asp/pART2-abc, asp/pART2-mfs, asp/pART2-mb1, asp/pART2-mb2, asp/pART2-AmceG, asp/pART2-AyrbE4A and Asp/pART2-AyrbE4B.
Example 3
Analysis of the ability of the control strains pART2 to dehydrogenate the steroid C1,2 of the engineering strains overexpressing abc, mfs, mb1, mb2
The abc, mfs, mb, mb2 over-expression engineering strain constructed in example 2 and the control strain pART2 are respectively picked from the inclined plane and inoculated into LB liquid culture medium containing 50 mug/mL kanamycin, and are cultured for 36h under shaking at 32 ℃ and 160r/min, and are transferred into a 250mL triangular flask containing 50mL of fresh culture medium with a certain inoculum size, and the initial OD is obtained 600 Adjusting the value to 0.2, performing shake culture at 32 ℃ at 160r/min to logarithmic growth phase, respectively adding a substrate Cortisone Acetate (CA) with a final concentration of 0.1g/L to induce the production of C1, 2-site dehydrogenase, and performing shake culture at 32 ℃ at 160r/min for 18h to the logarithmic middle and later stages of each strain. Centrifuging the culture solution at 4deg.C and 6000r/min for 10min, and pre-cooling the collected thallus with 0.1M KH of pH 7.2 2 PO 4 Washing 2 times with NaOH solution (PBS buffer solution), and suspending the thallus in a proper amount of PBS buffer solution to prepare resting cells.
30mL of the transformation system was prepared using resting cells as described above:
conversion system I: thallus OD 600 2.0, the concentration of the substrate CA is 6g/L, and 0% ethanol is used for assisting dissolution; the transformation process curve is shown in figure 3;
conversion system II: thallus OD 600 2.0, the concentration of the substrate CA is 6g/L,8% ethanol is used for assisting dissolution; the transformation process curve is shown in fig. 4;
conversion system III: thallus OD 600 2.0, the concentration of the substrate CA is 15g/L,8% ethanol is used for assisting dissolution; the transformation process curve is shown in FIG. 5;
conversion system IV: thallus OD 600 2.0, the concentration of the substrate CA is 90g/L,8% ethanol is used for assisting dissolution; the transformation process curve is shown in FIG. 6.
The concentration of the product Prednisone Acetate (PA) was determined by sampling at regular time and shaking at 34℃and 180r/min for conversion. Adding 0.8mL of ethyl acetate into 0.4mL of the sample each time to terminate the reaction, performing ultrasonic extraction for more than 10min, centrifuging for 10min at 12000r/min, sucking 100 mu L of supernatant into a new 1.5mL centrifuge tube, volatilizing overnight in a fume hood, re-dissolving with 1mL of mobile phase, and determining the conversion rate of the substrate CA and the yield of the product PA by an HPLC method.
The HPLC detection conditions were:
high performance liquid chromatograph: agilent 1100Series LC (G1314 Pump, G1322ADEGASSER G1314VWD detector, 10 μLAN injector, HP ChemStation);
chromatographic column: kromasil 100-5SIL 250 mm. Times.4.6 mm. Times.5 μm;
mobile phase: dichloromethane, diethyl ether and methanol (volume ratio of 86:12:2), and filtering with a microporous filter membrane of 0.45 μm;
flow rate: 1mL/min;
column temperature: 30 ℃;
a detector: UVDetector, wavelength: 240nm.
Sample injection amount: 20 mu L
Samples were taken at different times during the conversion process, the results of which are shown in table 1.
TABLE 1 comparison of the conversion results of Transporter overexpression engineering Strain to control Strain to cortisone acetate to prednisone acetate
As can be seen from Table 1, in the transformation system I, the concentration of PA generated by transforming CA by the abc and mb1 over-expression engineering bacteria is higher than that of the control strain pART2, and is respectively improved by 10.26% and 4.14%; in the transformation system II, the concentration of the PA generated by transforming CA by the abc and mb1 over-expression engineering bacteria is higher than that of the control strain pART2, and the concentration is respectively improved by 4.40 percent and 3.48 percent; in the transformation system III, the concentrations of PA generated by transforming CA by the abc, mfs, mb and mb2 over-expression engineering bacteria are obviously higher than those of the control strain pART2, and the concentration is abc (19.70%) > mb1 (6.29%) > mb2 (5.58%) > mfs (1.42%) in sequence from high to low; in the transformation system III, the concentration of PA generated by transforming CA with mfs, mb1 and mb2 over-expression engineering bacteria is obviously higher than that of the control strain pART2, and 21.79%, 51.74% and 50.67% are respectively improved.
Example 4
Analysis of the ability of the control Strain pART2 to dehydrogenate steroid C1,2 with AmceG, ayrbE4A, ayrbE B over-expressed engineering Strain
The AmceG, ayrbE4A, ayrbE B over-expression engineering strain and the control strain pART2 constructed in example 2 are respectively picked from the inclined plane, inoculated into LB liquid culture medium containing 50 mug/mL kanamycin, cultured for 36h at 32 ℃ under 160r/min in a shaking way, transferred into a 250mL triangular flask containing 50mL of fresh culture medium with a certain inoculum size, and the initial OD 600 Adjusting the value to 0.2, performing shake culture at 32 ℃ at 160r/min to logarithmic growth phase, respectively adding a substrate Cortisone Acetate (CA) with a final concentration of 0.1g/L to induce the production of C1, 2-site dehydrogenase, and performing shake culture at 32 ℃ at 160r/min for 18h to the logarithmic middle and later stages of each strain. Centrifuging the culture solution at 4deg.C and 6000r/min for 10min, and pre-cooling the collected thallus with 0.1M KH of pH 7.2 2 PO 4 Washing 2 times with NaOH solution (PBS buffer solution), and suspending the thallus in a proper amount of PBS buffer solution to prepare resting cells.
30mL of the transformation system was prepared using resting cells as described above: thallus OD 600 2.0, the concentration of the substrate CA is 15g/L,8% ethanol is used for assisting dissolution; the concentration of the product Prednisone Acetate (PA) was determined by sampling at regular time and shaking at 34℃and 180r/min for conversion. 0.4mL of each sample was addedTerminating the reaction with 0.8mL ethyl acetate, ultrasonically extracting for more than 10min, centrifuging for 10min at 12000r/min, sucking 100 mu L supernatant into a new 1.5mL centrifuge tube, volatilizing overnight in a fume hood, re-dissolving with 1mL mobile phase, and determining the conversion rate of the substrate CA by an HPLC method to obtain the product PA.
HPLC detection conditions were the same as in example 3.
Samples were taken at various times during the transformation process, the transformation process curves are shown in FIG. 7, and the results are shown in Table 2.
TABLE 2 comparison of the conversion results of Transporter AMceG, AYrbE4A, AYrbE B over-expressed engineering strains and control strains to prednisone acetate
As shown in Table 2, in the transformation system with the substrate CA concentration of 15g/L and the ethanol concentration of 8%, the concentration of the produced PA by transforming CA with the AmceG and AyrbE4A, ayrbE B over-expression engineering bacteria is obviously higher than that of the control strain pART2, and the concentration of the produced PA is sequentially from high to low, amceG (13.52%) > AyrbE4A (8.53%) > AyrbE4B (4.26%).
Example 5
Construction of Arthrobacter simplex gene engineering strain for coexpression of transport protein and heat shock protein genes
5.1 construction of recombinant plasmid pART2-groEL
Using G + Bacterial genomic DNA extraction kit (Tiangen Biochemical technology Co., ltd.) total DNA of Arthrobacter simple CPCC 140451 was extracted. And amplifying groEL genes by taking the extracted total DNA as a template. The primer sequences and the corresponding codes of the sequence table are shown in the following table:
note that: underline indicates the cleavage site
The PCR reaction system is shown in the following table (50. Mu.L):
the PCR reaction conditions were: the product was subjected to agarose gel electrophoresis to determine whether the fragment size was consistent after the PCR was completed, at 95℃for 2min,95℃for 20s,55-65℃for 20s,72℃for 2min, and 35 cycles for 72℃for 5 min.
The PCR product was purified using a DNA purification kit (Shanghai Biotechnology service Co., ltd.) and then digested, and the reaction system was as follows:
the reaction was carried out in a bath at 37℃for 2h.
And (3) respectively carrying out a ligation reaction (16 ℃ for 12 hours) on the prepared target fragments and the vector plasmid pART2 subjected to enzyme digestion and purification according to the proportion of 1:3, respectively converting the ligation products into escherichia coli DH5 alpha, and screening to obtain the recombinant plasmid pART2-groEL.
5.2 construction of recombinant plasmid pART2-groEL-abc
The recombinant plasmid pART2-abc is used as a template to amplify a promoter and an abc gene, and the corresponding codes of a primer sequence and a sequence table of the promoter and the abc gene are shown in the following table:
the PCR reaction system is shown in the following table (50. Mu.L):
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the PCR reaction conditions were: the products were subjected to agarose gel electrophoresis to determine whether the fragment size was consistent after the PCR was completed, at 95℃for 2min,95℃for 20s,55℃for 20s,72℃for 2min, and 35 cycles for 72℃for 5 min.
The PCR product was purified using a DNA purification kit (Shanghai Biotechnology services Co., ltd.). The recombinant plasmid pART2-groEL was subjected to cleavage, and the reaction system was as follows:
the reaction was carried out in a bath at 37℃for 2h.
And (3) respectively carrying out a ligation reaction (55 ℃ for 15 min) on the prepared target fragments and the vector plasmid pART2-groEL subjected to enzyme digestion and purification by using a Gibson assembly method, respectively converting the ligation products into escherichia coli DH5 alpha, and screening to obtain the recombinant plasmid pART2-groEL-abc.
5.3 construction of Co-expressed Arthrobacter Simplex engineering Strain Asp/pART2-groEL-abc
(1) Preparation of Arthrobacter simple competent cells
The preparation of Arthrobacter simple competence was as described in example 2.
(2) Recombinant plasmid electric shock transformation and verification
Taking 120 mu L of Arthrobacter simplicissimus competent cells, adding 200ng of constructed recombinant plasmid pART2-groEL-abc into a 1.5mL centrifuge tube, uniformly mixing, and transferring to a precooled electric pulse cup for ice bath for 3min; turning on an electric pulse instrument, and converting 2.1kV electric shock; immediately after electric excitation, 0.8mL of sterile resuscitating medium (LB liquid medium containing 0.5moL/L sorbitol) was added into the electric pulse cup, after mixing, the mixture was slowly cultured by shaking at 32℃for 11 hours, and then the mixture was spread on a selection plate containing 50. Mu.g/mL kanamycin, and the mixture was cultured upside down at 32℃for 72 hours to pick up transformants. As shown in FIG. 8, the PCR amplification shows a band around 2600bp, which is consistent with the length 2609bp of the target fragment "groEL (1632 bp) +promoter (218 bp) +abc (759 bp)"; the plasmid is digested with EcoR I and Xba I to form bands at 4000bp and 3200bp, which are respectively consistent with the sizes of the vector (3994 bp) and the fragment (3205 bp), which shows that the engineering strain successfully expresses groEL and abc genes. It was sent to the gold only biotechnology limited company for further sequencing to be verified, and the engineering strain which was verified to be correct was named Asp/pART2-groEL-abc.
Example 6
Analysis of the ability of the control Strain pART2 to co-express the steroid C1,2 of the Arthrobacter similis engineering Strain with groEL-abc to dehydrogenation reaction
The groEL-abc co-expression engineering strain constructed in example 5 and the control strain pART2 were respectively picked from the slant, inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, shake-cultured at 32℃for 36h at 160r/min, and transferred into a 250mL Erlenmeyer flask containing 50mL of the fresh medium at a fixed inoculum size, and the original OD 600 Adjusting the value to 0.2, performing shake culture at 32 ℃ at 160r/min to logarithmic growth phase, respectively adding a substrate Cortisone Acetate (CA) with a final concentration of 0.1g/L to induce the production of C1, 2-site dehydrogenase, and performing shake culture at 32 ℃ at 160r/min for 18h to the logarithmic middle and later stages of each strain. Centrifuging the culture solution at 4deg.C and 6000r/min for 10min, and pre-cooling the collected thallus with 0.1M KH of pH 7.2 2 PO 4 Washing 2 times with NaOH solution (PBS buffer solution), and suspending the thallus in a proper amount of PBS buffer solution to prepare resting cells.
30mL of the transformation system was prepared using resting cells as described above:
conversion system I: thallus OD 600 2.0, the concentration of the substrate CA is 6g/L,8% ethanol is used for assisting dissolution; the transformation process curve is shown in FIG. 9;
conversion system II: thallus OD 600 2.0, the concentration of the substrate CA is 15g/L,8% ethanol is used for assisting dissolution; the transformation process curve is shown in FIG. 10;
conversion system III: thallus OD 600 2.0, the concentration of the substrate CA is 90g/L,8% ethanol is used for assisting dissolution; the transformation process curve is shown in FIG. 11.
The concentration of the product Prednisone Acetate (PA) was determined by sampling at regular time and shaking at 34℃and 180r/min for conversion. Adding 0.8mL of ethyl acetate into 0.4mL of the sample each time to terminate the reaction, performing ultrasonic extraction for more than 10min, centrifuging for 10min at 12000r/min, sucking 100 mu L of supernatant into a new 1.5mL centrifuge tube, volatilizing overnight in a fume hood, re-dissolving with 1mL of mobile phase, and determining the conversion rate of the substrate CA and the yield of the product PA by an HPLC method.
HPLC detection conditions were the same as in example 3.
Samples were taken at different times during the conversion process, the results of which are shown in table 3.
TABLE 3 comparison of the conversion results of prednisone acetate from the conversion of cortisone acetate by groEL-abc Co-expression engineering Strain with control Strain
As shown in Table 3, in the transformation system I, the PA yield of the abc and groEL co-expression engineering bacteria reaches the maximum when the engineering bacteria are transformed for 6 hours, and is 5.58g/L, which is improved by 2.20% compared with the control strain (5.46 g/L); in the transformation system II, the PA production amount of the abc and groEL co-expression engineering bacteria reaches the maximum when the transformation is carried out for 36h, the PA production amount is 12.58g/L, the PA production amount is 27.72% higher than that of a control strain (9.85 g/L), the PA production amount is higher than that of the abc over-expression engineering bacteria (18.27%) in the transformation system III of the embodiment 3, and the transformation time is shortened from 48h to 36h; in the transformation system III, the PA production amount of the abc and groEL co-expression engineering bacteria reaches the maximum when the abc and groEL co-expression engineering bacteria are transformed for 108 hours, and is 22.19g/L, and the PA production amount is improved by 19.11 percent compared with that of a control strain (18.63 g/L).
The above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Accordingly, the protection scope of this patent shall be subject to the appended claims.

Claims (10)

1. A steroid C1,2 dehydrogenation reaction engineering strain with excellent transport and reaction activity is characterized in that: the genetically engineered bacterium is obtained by using Arthrobacter simplex (Arthrobacter simplex) CPCC 140451 as a host cell and over-expressing any one of genes shown in a sequence table SEQ ID No. 4.
2. The steroid C1,2 dehydrogenation reaction engineering strain having excellent transport and reactivity according to claim 1, characterized in that: the nucleotide sequence is shown as SEQ ID No.4 to express the transporter as MB2.
3. The steroid C1,2 dehydrogenation reaction engineering strain having excellent transport and reactivity according to claim 1, characterized in that: the genetic engineering bacteria take pART2 plasmid as an expression vector.
4. A method for constructing a steroid C1,2 dehydrogenation reaction engineering strain having excellent transport and reactivity according to claim 1, characterized by: the method comprises the following steps:
the method comprises the steps of using a simple arthrobacter CPCC 140451 genome as a template, and obtaining a transporter coding gene in the strain through PCR amplification, wherein the gene is any gene shown in SEQ ID No. 4;
secondly, the transporter coding genes are respectively connected to promoters of an escherichia coli-arthrobacter shuttle plasmid pART2 by using a genetic engineering means, and then transformed into escherichia coli DH5 alpha for replication;
thirdly, extracting recombinant plasmids, and respectively electrically transforming the recombinant plasmids into Arthrobacter simplex CPCC 140451 for overexpression.
5. A method for catalyzing cortisone acetate to generate C1, 2-position dehydrogenation reaction, which is characterized by comprising the following steps: use of a Arthrobacter simplex gene engineering bacterium having strong transport ability according to any one of claims 1 to 4.
6. An engineered strain of Arthrobacter simplex with strong substrate transport and C1,2 reaction capability, which is characterized in that: the genetically engineered bacterium is obtained by co-expressing a gene shown in a sequence table SEQ ID No.4 and a heat shock protein coding gene shown in a sequence table SEQ ID No.8 by taking Arthrobacter simplex (Arthrobacter simplex) CPCC 140451 as a host cell and pART2 plasmid as an expression vector.
7. A method for constructing a steroid C1,2 dehydrogenation reaction engineering strain having excellent transport and reactivity according to claim 6, characterized by: the method comprises the following steps:
the method comprises the steps of using a simple arthrobacter CPCC 140451 genome as a template, and obtaining a transporter coding gene and a heat shock protein coding gene in the strain through PCR amplification, wherein the transporter coding gene is any gene shown in SEQ ID No.4, and the heat shock protein coding gene is a gene shown in SEQ ID No. 8;
secondly, connecting the coding genes of the transport protein and the heat shock protein in series by utilizing a genetic engineering means, and then, transferring the genes into escherichia coli DH5 alpha for replication after connecting the genes to a promoter of an escherichia coli-arthrobacter shuttle plasmid pART 2;
thirdly, extracting recombinant plasmids, and respectively electrically transforming the recombinant plasmids into Arthrobacter simplex CPCC 140451 for overexpression.
8. A method for catalyzing cortisone acetate to generate C1, 2-position dehydrogenation reaction, which is characterized by comprising the following steps: use of the Arthrobacter simplex gene engineering bacterium having strong transport ability and stress tolerance according to any one of claims 6 to 7 for substrate conversion of cortisone acetate.
9. The method of catalyzing the C1,2 dehydrogenation reaction of cortisone acetate according to claim 5 or claim 8, wherein: the concentration of cortisone acetate as substrate in the conversion system is 0-120g/L, preferably 6-90g/L, more preferably 10-30g/L.
10. A method of catalyzing the C1,2 dehydrogenation of cortisone acetate according to claim 5 or claim 8, characterized in that: the ethanol concentration in the conversion system is 4-15%, preferably 6-10%.
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