CN114395519A - Recombinant bacterium and method for expressing ketoreductase - Google Patents

Recombinant bacterium and method for expressing ketoreductase Download PDF

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Publication number
CN114395519A
CN114395519A CN202111649747.6A CN202111649747A CN114395519A CN 114395519 A CN114395519 A CN 114395519A CN 202111649747 A CN202111649747 A CN 202111649747A CN 114395519 A CN114395519 A CN 114395519A
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ketoreductase
present application
recombinant
gly
ala
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倪晶霞
尚永崇
崔俊杰
孙丰来
徐幸福
朱景仰
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Shanghai SynTheAll Pharmaceutical Co Ltd
Shanghai STA Pharmaceutical R&D Ltd
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Shanghai SynTheAll Pharmaceutical Co Ltd
Shanghai STA Pharmaceutical R&D Ltd
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    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

Abstract

The application discloses a recombinant bacterium and a method for expressing ketoreductase. The recombinant strain comprises a molecular chaperone vector and expresses ketoreductase, and the molecular chaperone vector comprises a pGro7 vector.

Description

Recombinant bacterium and method for expressing ketoreductase
Technical Field
The application relates to the technical field of enzymes, in particular to a recombinant bacterium and a method for expressing ketoreductase.
Background
Coli as the most common prokaryotic expression system for efficiently expressing heterologous proteins has the advantages of simple structure, high growth speed, easy culture, clear genetic background, convenient gene operation and the like. Although E.coli has numerous advantages, not every gene can be efficiently expressed therein.
This is due to the unique structure of each gene, the stability and translation rate of the mRNA, the ease of protein folding, the degradation of proteins by host cell proteases, differences in codon usage of foreign genes and E.coli, and the potential toxicity of proteins to the host.
Therefore, the expression of heterologous proteins using E.coli expression systems is more or less difficult in practical applications, and most often the target protein forms insoluble inclusion bodies, which brings many inconveniences to subsequent research. Inclusion formation may be due to the failure of nascent peptide chains to form disulfide bonds in the correct position, leading to disulfide mismatch and thus misfolding [ ANNA MITRAKI, BENTLEY FANE, [ CAMERON HAASE-PETJINGELL et al.Global rendering of protein folding defects and inclusion body formation [ J ]. science.1991: 253(5015): 54-58.]. The molecular chaperone can help the protein to be correctly folded, improve the soluble expression of the protein and reduce the formation of inclusion bodies. Pfeffer et al [ Jan Pfeffer, Monika Rusnak, Carl-Erik Hansen, et al.functional expression of lipase A from Candida antarctica in Escherichia coli-A prediction for high-throughput screening and direct evolution [ J ]. Journal of Molecular Catalysis B: enzymic 45 (2007): 62-67 ] the expression of the molecular chaperone protein while the target protein is expressed in the escherichia coli improves the soluble expression ratio of the target protein. The method of co-expressing with molecular chaperone to raise the soluble target protein is becoming important for researchers.
Chinese patent application CN110499312A discloses a label and a method for improving the soluble expression of enzyme, and Chinese patent application CN112812191A discloses a fusion label for improving the soluble expression of enzyme and application thereof. However, these general strategies are not effective against all enzymes. The art has not disclosed a proven viable method for increasing soluble expression of ketoreductases.
There remains a need in the art to find a way to increase the soluble expression of ketoreductases.
Disclosure of Invention
To solve the above technical problems, one aspect of the present application provides a recombinant bacterium comprising a chaperone vector including a pGro7 vector and expressing a ketoreductase.
In another aspect of the invention, there is provided a method of expressing a ketoreductase enzyme, the method comprising:
a. providing a recombinant bacterium comprising a chaperone vector including a pGro7 vector and expressing a ketoreductase;
b. culturing the recombinant strain; and
c. inducing the recombinant bacteria at a lower temperature to express the ketoreductase.
Drawings
The present application is described in more detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a gel electrophoresis diagram showing the protein expression profile during fermentation at 20 ℃ for 16h with 0.1mM IPTG added when a ketoreductase-pGro 7 recombinant strain according to one embodiment of the present application was cultured at 37 ℃ until OD reached 0.6, wherein: m is a protein molecular weight standard Marker, 1 is a whole cell after 0h of induction, 2 is a whole cell after 16h of induction, 3 is a supernatant after 16h of induction, and 4 is a precipitate after 16h of induction.
FIG. 2 is a gel electrophoresis diagram showing the protein expression profile during 12h fermentation at 20 ℃ with addition of 0.1mM IPTG and addition of 0.2mg/mL arabinose when the recombinant strain pGro7, a ketoreductase according to one embodiment of the present application, is cultured at 37 ℃ until OD reaches 0.3, and then cultured again until OD reaches 0.6, wherein: m is a protein molecular weight standard Marker, 1-5 are whole cells for inducing expression for 0h, 3h, 6h, 9h and 12h respectively, 6 is supernatant after 12h of induction, and 7 is precipitate after 12h of induction.
FIG. 3 is a gel electrophoresis diagram showing the protein expression profile during 16h fermentation at 25 ℃ with addition of 0.2mg/mL arabinose when the recombinant strain pGro7, a ketoreductase according to one embodiment of the present application, was cultured at 37 ℃ until OD reached 0.3, and 0.1mM IPTG was added until OD reached 0.6, wherein: m is a protein molecular weight standard Marker, 1-5 are whole cells for inducing expression for 0h, 4h, 8h, 12h and 16h respectively, 6 is supernatant after inducing for 16h, and 7 is precipitate after inducing for 16 h.
FIG. 4 is a gel electrophoresis diagram showing the protein expression profile during fermentation at 20 ℃ for 17h with 0.1mM IPTG added when the ketoreductase- -pKJE7 recombinant strain was cultured at 37 ℃ until OD reached 0.3 and with 0.2mg/mL arabinose added when OD reached 0.6, wherein: m is a protein molecular weight standard Marker, 1 is a whole cell after 0h of induction, 2 is a whole cell after 17h of induction, 3 is a supernatant after 17h of induction, and 4 is a precipitate after 17h of induction.
FIG. 5 is a gel electrophoresis diagram showing the protein expression profile during the fermentation of ketoreductase- -pG-Tf2 recombinant bacteria at 37 ℃ to OD 0.3 with 0.2mg/mL arabinose, 0.1mM IPTG to OD 0.6, and 20 ℃ for 17h, wherein: m is a protein molecular weight standard Marker, 1 is a whole cell after 0h of induction, 2 is a whole cell after 17h of induction, 3 is a supernatant after 17h of induction, and 4 is a precipitate after 17h of induction.
FIG. 6 is a gel electrophoresis diagram showing the protein expression profile during 18h fermentation at 20 ℃ with 0.1mM IPTG added when the ketoreductase- -pG-KJE8 recombinant strain was cultured at 37 ℃ until OD reached 0.3 and with 0.2mg/mL arabinose added to continue the culture until OD reached 0.6, wherein: m is a protein molecular weight standard Marker, 1 is a whole cell after 0h of induction, 2 is a whole cell after 18h of induction, 3 is a supernatant after 18h of induction, and 4 is a precipitate after 18h of induction.
FIG. 7 is a gel electrophoresis diagram showing the protein expression profile during 18h fermentation at 20 ℃ with the ketoreductase- -pTf16 recombinant bacteria cultured at 37 ℃ to an OD of 0.3 with the addition of 0.2mg/mL arabinose with the addition of 0.1mM IPTG with the addition of 0.6 OD, and further cultured with the addition of 20 ℃ OD: m is a protein molecular weight standard Marker, 1 is a whole cell after 0h of induction, 2 is a whole cell after 18h of induction, 3 is a supernatant after 18h of induction, and 4 is a precipitate after 18h of induction.
FIG. 8 is a high performance gas chromatography spectrum of ethyl 4-chloro-3-hydroxy-butyrate after conversion by method (1) of example 3. the peak at the position of t-7.687 is the target compound of 4-chloro-3-hydroxy-ethyl butyrate.
Detailed Description
The present application relates to a recombinant bacterium comprising a chaperone vector including a pGro7 vector and expressing a ketoreductase. In one embodiment of the present application, the pGro7 vector expresses pGro7 fusion tag. Recombinant bacteria expressing ketoreductase are commercially available. In one embodiment of the present application, recombinant bacteria expressing ketoreductase are obtained by transfection of a plasmid encoding ketoreductase. In one embodiment of the present application, the ketoreductase is from Rhodococcus ruber strain SD 3. In a preferred embodiment of the present application, the ketoreductase has an amino acid sequence as set forth in SEQ ID NO: 2, respectively. In a preferred embodiment of the present application, the nucleotide sequence encoding the ketoreductase enzyme is as set forth in SEQ ID NO: 1 is shown. In the present application, the recombinant bacterium may be any host bacterium suitable for expressing ketoreductase. In one embodiment of the present application, the recombinant bacterium comprises escherichia coli. In a preferred embodiment of the present application, the E.coli comprises E.coli BL21(DE) 3. In a preferred embodiment of the present application, E.coli comprises E.coli BL21(DE)3 Rhodococcus ruber strain SD 3.
The application also relates to a method of expressing ketoreductase enzymes. The method comprises the following steps: provided is a recombinant bacterium comprising a chaperone vector including pGro7 vector and expressing ketoreductase. In one embodiment of the present application, the recombinant bacterium is the aforementioned recombinant bacterium.
The methods of expressing ketoreductase enzymes of the present application further comprise: and (5) culturing the recombinant bacteria. In the present application, the recombinant bacteria are cultured in any suitable medium known in the art. In one embodiment of the present application, the culture medium comprises M9 culture medium.
The methods of expressing ketoreductase enzymes of the present application further comprise: inducing the recombinant bacteria at a lower temperature to express the ketoreductase. In the present application, lower temperature means a temperature lower than the optimal growth temperature of the recombinant bacteria. For example, for E.coli, lower temperatures refer to temperatures below 37 ℃. In one embodiment of the present application, the lower temperature is 10-35 ℃. In a preferred embodiment of the present application, the lower temperature is 15-30 ℃. In a preferred embodiment of the present application, the lower temperature is 20-25 ℃. In one embodiment of the present application, the induction lasts 5-50 hours. In a preferred embodiment of the present application, the induction lasts 10-20 hours. In a preferred embodiment of the present application, the induction lasts 12-16 hours. In the present application, the recombinant bacteria may be induced to express the ketoreductase by any suitable means. In one embodiment of the present application, the gene encoding the ketoreductase is on an inducible expression vector. In one embodiment of the present application, inducing comprises using an inducing agent. In the present application, the inducer can be any inducer that induces the expression of the ketoreductase enzyme. In a preferred embodiment of the present application, the inducing agent comprises one or more of the following: arabinose and IPTG. In one embodiment of the present application, inducing comprises adding an inducer at a specific culturing stage. In one embodiment of the present application, inducing comprises: (1) adding arabinose when OD of cultivation reaches 0.2-0.4, preferably 0.25-0.35, more preferably about 0.3; and (2) when the culture OD reaches 0.5 to 0.7, preferably 0.55 to 0.65, more preferably about 0.6, IPTG is added. In a preferred embodiment of the present application, (1) comprises the addition of 0.1-0.5mg/mL arabinose. In a preferred embodiment of the present application, (1) comprises the addition of 0.15-0.3mg/mL arabinose. In a preferred embodiment of the present application, (1) comprises adding about 0.2mg/mL arabinose. In a preferred embodiment of the present application, (2) comprises the addition of 0.05-0.3mmol/L of IPTG. In a preferred embodiment of the present application, (2) comprises adding 0.08-0.15mmol/L of IPTG. In a preferred embodiment of the present application, (2) comprises adding about 0.1mmol/L of IPTG. In one embodiment of the present application, the inducing may comprise a plurality of inducing processes at different temperatures. In one embodiment of the present application, the addition of (1) and (2) is performed at a first temperature, and the temperature is adjusted to a second temperature after the addition of (2) is completed. In one embodiment of the present application, the first temperature is higher than the second temperature. In one embodiment of the present application, the first temperature is an optimal growth temperature for the recombinant bacteria, for example about 37 ℃. In one embodiment of the present application, the second temperature is the lower temperature described above. In a preferred embodiment of the present application, the addition of (1) and (2) is carried out at about 37 ℃ and the temperature is reduced to 20-25 ℃ after the addition of (2) is completed and the induction is continued for 12-16 hours.
The technical solutions of the present application will be described in detail and fully with reference to the accompanying drawings, and it should be understood that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without any inventive step are within the scope of protection of the present application.
Experimental Material
Coli BL21(DE)3 competent cells: purchased from heaven root, cat # CB 105. The ketoreductase gene is derived from Rhodococcus ruber (Rhodococcus ruber) SD3, and the specific gene sequence is shown in appendix SEQ ID NO: 1.
LB liquid Medium (g/L): tryptone: 10, yeast extract: 5, sodium chloride: 10.
an OD detection method comprises the following steps: and diluting the bacterial liquid by a proper time, and measuring the data at the absorbance 600 of an ultraviolet spectrophotometer.
pGro7 plasmid: purchased from Takara, Cat. # 9122.
pG-KJE8 plasmid: purchased from Takara, Cat. # 9121.
pKJE7 plasmid: purchased from Takara, Cat. # 9123.
pG-Tf2 plasmid: purchased from Takara, Cat. # 9124.
pTf16 plasmid: purchased from Takara, Cat. # 9125.
Marker: buy bi yun tian, cat.
34mg/mL chloramphenicol: 1.7g of chloramphenicol was weighed out and dissolved in 40mL of absolute ethanol to a constant volume of 50 mL.
LB solid Medium (g/L): tryptone: 10, yeast extract: 5, sodium chloride: 10, agar: 20.
m9 Medium (g/L): k2HPO4：12.5，KH2PO4: 6.25, yeast extract: 6.67 (NH)4)2SO4: 1.76, glycerin: 1, PPG2000 (anti-foam): 0.2, MgSO4：0.26，CaCl2: 0.023, trace elements: 1.
200mM phosphate buffer (pH 7.0): 35.81g of Na were weighed2HPO4·12H2Adding deionized water to the solution to reach the volume of 500ml, and weighing 7.8g of NaH2PO4·2H 20, adding deionized water to a constant volume of 500ml, mixing the two at a ratio of 62: 38, and adjusting the pH to 7.0.
SDS PAGE gel electrophoresis method:
(1) appropriate amount of 1 xSDS-PAGE Hepes electrophoresis buffer (20 xBeyogel)TMDiluting Plus SDS-PAGE Hepes electrophoresis buffer solution by adding ultrapure water; the internal liquid needs to be prepared as required; the external liquid can be reused for about 10 times).
(2) And taking out the rubber plate, inwards fixing the short plate in the rubber plate clamping groove, and then placing the rubber plate clamping groove on the electrophoresis tank bracket.
(3) Pouring a proper amount of prepared buffer solution into the electrophoresis tank, wherein the buffer solution in the inner tank is required to be above the short rubber plate, and the amount of the buffer solution in the outer tank can be added according to the indication on the electrophoresis tank.
(4) Carefully remove the comb.
(5) After 1mL of the bacterial liquid is centrifuged at 12000rpm for 3min, the cell is retained by washing with 0.9% NaCl and then resuspended in 1mL of 0.9% NaCl to obtain a whole cell sample. Centrifuging 1mL of bacterial solution at 12000rpm for 3min, washing and centrifuging with 0.9% NaCl, reserving cells, then re-suspending the cells with 1mL of 0.9% NaCl, carrying out ultrasonic disruption (200w, disruption for 1s, stopping for 2s, and 10min), centrifuging at 12000rpm for 3min, sucking the supernatant for storage, obtaining a supernatant sample, washing the disruption solution precipitate left by centrifugation with 1mL of 0.9% NaCl, centrifuging, and finally re-suspending the precipitate with 1mL of 0.9% NaCl, thus obtaining a precipitate sample.
(6) Sampling; 10 mu L of sample is taken; marker: 6 μ L.
(7) After the sample is added, the cover of the electrophoresis tank is covered, and the electrophoresis apparatus is connected (red to red, black to black).
(8) Electrophoresis is started, and electrophoresis program can be carried out according to the indication of the rubber plate.
(9) And stopping electrophoresis when the bromophenol blue indicator migrates to a position 1-2 cm away from the front edge.
(10) And after electrophoresis is finished, closing the power supply and taking out the rubber plate.
(11) And slightly prying the rubber plate, and transferring the glue into the developing solution until the developing solution is submerged by the glue.
(12) Placing the seeds on a shaking bed, and setting the procedures as follows: 100r/min, 15 min.
(13) The color developing solution is recovered, and the gum is slightly washed twice by a proper amount of clear water.
(14) And photographing and processing the photos.
Name (R) Manufacturer(s) Goods number/specification
MgCl2 SHANGHAI TITAN TECHNOLOGY Co.,Ltd. AR
CaCl2 Shanghai Aibi chemical reagent Co., Ltd AR
NaCl SHANGHAI EXPERIMENTAL REAGENT Co.,Ltd. AR
Glycerol SINOPHARM CHEMICAL REAGENT Co.,Ltd. 625GM
Kanamycin SANGON BIOTECH (SHANGHAI) Co.,Ltd. KB0286
Chloromycetin SANGON BIOTECH (SHANGHAI) Co.,Ltd. CB0118
IPTG Beyotime ST098
Arabinose Beyotime ST1420
4-Chloroacetoacetic acid ethyl ester Sigma-Aldrich GC
Isopropanol (I-propanol) Sigma-Aldrich LC
NADP Sigma-Aldrich 25g
Gas chromatography SHIMADZU GC-2030
Centrifugal machine Shanghai Congress Instrument Co Ltd MD80R
Fermentation tank Saisi Boehu PROLAB type D
Super clean bench Taishida Aeolus V6
Metal bath Saimei fly CRS10
Constant temperature box Wiegand technology (Beijing) Ltd WH-05
Culture box Shanghai Congress Instrument Co Ltd ISF-1-XSMX1503
Crushing machine NINGBO SCIENTZ BIOTECHNOLOGY Co.,Ltd. Scientz-950E
Example 1
Construction of recombinant bacterium
(1) Preparation of competent cells
Selecting ketoreductase single strain, adding into 10mL LB liquid medium containing kanamycin resistance with final concentration of 50ug/mL, culturing at 37 deg.C for 12 hr, and culturing ketoreductase strainInoculating into LB liquid medium, culturing at 37 deg.C until OD is 0.4-0.6, and placing on ice for 20 min. The solution is divided into 50mL liquid and centrifuged for 15min at 4000rpm and 4 ℃ to leave precipitate. With 100mM MgCl2Resuspend, centrifuge at 3000rpm for 15min at 4 ℃ and leave the pellet. Continued with 100mM CaCl2Resuspend, ice-bath for 20min, 3000rpm, 4 ℃ centrifugation for 15 min. First using 85mM CaCl2Washing the cells with 15% glycerol, centrifuging at 2500rpm at 4 ℃ for 15min, and finally adding 85mM CaCl2The 15% glycerol was resuspended and ketoreductase competent cells were prepared.
(2) Transformation of
50ul of ketoreductase competent cells and 5ul of pGro7, pG-KJE8, pKJE7, pG-Tf2 or pTf16 plasmids were pipetted into a pre-cooled EP tube with a sterile pipette in a clean bench, ice-cooled for 30min, heat-shocked with a metal bath at 42 ℃ for 2min and immediately transferred into the ice-bath for 15min, and then 700ul of LB medium was added and shake-cultured at 37 ℃ in a thermostat for 1 h. 200ul of the liquid was pipetted and spread on LB solid medium containing antibiotics, and placed in an incubator at 37 ℃ overnight for culture. Respectively obtaining LB solid culture medium of single colony containing ketoreductase-pGro 7 recombinant bacteria, ketoreductase-pG-KJE 8 recombinant bacteria, ketoreductase-pKJE 7 recombinant bacteria, ketoreductase-pG-Tf 2 recombinant bacteria and ketoreductase-pTf 16 recombinant bacteria.
Example 2
Ketoreductase- -low temperature induced expression of pGro7 recombinant bacterium
The ketoreductase-pGro 7 recombinant strain obtained in example 1 was picked up and dropped into LB liquid medium, activated overnight at 37 ℃, the seed solution was inoculated into M9 medium in an amount of 1% of the uptake, IPTG was added to the final concentration of 0.1mM when OD reached 0.6, and the mixture was fermented at 20 ℃ for 16 hours, followed by centrifugation to collect the strain and disrupt the cells. The cells were disrupted for 15min and inclusion bodies were collected. The molecular weight of the target protein is 35.2 KDa. The expression results are shown in FIG. 1. The content of the target protein in the supernatant is low, and the soluble expression is poor.
Example 3
ketoreductase-pGro 7 recombinant strain low-temperature induced expression (II)
(1) Culturing at 20 deg.C and inducing for 12 hr
A single strain of the ketoreductase-pGro 7 recombinant strain obtained in example 1 was picked up and dropped into LB liquid medium, activated overnight at 37 ℃, the seed solution was inoculated into M9 medium in an amount of 1% of the uptake, arabinose was added to the medium at a final concentration of 0.2mg/mL and the culture was continued at 37 ℃ until OD reached 0.3, IPTG was added to the medium at a final concentration of 0.1mM until OD reached 0.6, and the mixture was fermented at 20 ℃ for 12 hours, and then cells were collected by centrifugation and disrupted. The cells were disrupted for 15min and inclusion bodies were collected. The molecular weight of the target protein is 35.2 KDa. The expression results are shown in FIG. 2. The content of the target protein in the supernatant (namely the crude enzyme solution) is higher, and the soluble expression is better.
(2) Culturing at 25 deg.C and inducing for 16h
The ketoreductase-pGro 7 recombinant strain obtained in example 1 was picked up and dropped into LB liquid medium, activated overnight at 37 ℃, the seed solution was inoculated into M9 medium in an amount of 1% of the uptake, arabinose was added to the medium at a final concentration of 0.2mg/mL and the culture was continued at 37 ℃ until OD reached 0.3, IPTG was added to the medium at a final concentration of 0.1mM until OD reached 0.6, and the mixture was fermented at 25 ℃ for 16 hours, then cells were collected by centrifugation and disrupted. The cells were disrupted for 15min and inclusion bodies were collected. The molecular weight of the target protein is 35.2 KDa. The expression results are shown in FIG. 3. The content of the target protein in the supernatant is higher, and the soluble expression is better.
Example 4
Low-temperature induced expression of ketoreductase-pKJE 7 recombinant strain
A single strain of the ketoreductase- - -pKJE7 recombinant strain obtained in example 1 was picked up and dropped into LB liquid medium, activated overnight at 37 ℃, the seed solution was inoculated into M9 medium in an amount of 1% of the uptake, arabinose was added to the solution at a final concentration of 0.2mg/mL and the mixture was further cultured at 37 ℃ until the OD reached 0.3, IPTG was added to the solution at a final concentration of 0.1mM until the OD reached 0.6, and after fermentation at 25 ℃ for 17 hours, the strain and the cells were collected by centrifugation and disrupted. The cells were disrupted for 15min and inclusion bodies were collected. The molecular weight of the target protein is 35.2 KDa. The expression results are shown in FIG. 4. The overall solubility expression of the target protein is less.
Example 5
Low-temperature induced expression of ketoreductase-pG-Tf 2 recombinant bacteria
Selecting a single strain of the ketoreductase- - -pG-Tf2 recombinant strain obtained in example 1, dropping the single strain into an LB liquid culture medium, activating the strain overnight at 37 ℃, inoculating a seed solution into an M9 culture medium according to the suction quantity of 1%, culturing the strain at 37 ℃ until OD reaches 0.3, adding arabinose with the final concentration of 0.2mg/mL, continuing culturing the strain until OD reaches 0.6, adding IPTG with the final concentration of 0.1mM, fermenting the strain at 25 ℃ for 17 hours, and centrifugally collecting strains and broken cells. The cells were disrupted for 15min and inclusion bodies were collected. The molecular weight of the target protein is 35.2 KDa. The expression results are shown in FIG. 5. The overall solubility expression of the target protein is less.
Example 6
Low-temperature induced expression of ketoreductase-pG-KJE 8 recombinant bacteria
A single strain of the ketoreductase- - -pG-KJE8 recombinant strain obtained in example 1 was picked up and dropped into LB liquid medium, activated overnight at 37 ℃, the seed solution was inoculated into M9 medium in an amount of 1% of the uptake, arabinose was added to the solution at a final concentration of 0.2mg/mL and the mixture was further cultured at 37 ℃ until OD reached 0.3, IPTG was added to the solution at a final concentration of 0.1mM when OD reached 0.6, and after fermentation at 25 ℃ for 18 hours, the strain and disrupted cells were collected by centrifugation. The cells were disrupted for 15min and inclusion bodies were collected. The molecular weight of the target protein is 35.2 KDa. The expression results are shown in FIG. 6. The overall solubility expression of the target protein is less.
Example 7
ketoreductase-pTf 16 recombinant strain low-temperature induced expression
A single strain of the ketoreductase-pTf 16 recombinant strain obtained in example 1 was picked up and dropped into LB liquid medium, activated overnight at 37 ℃, the seed solution was inoculated into M9 medium in an amount of 1% by suction, cultured at 37 ℃ until OD reached 0.3, arabinose was added to the solution to a final concentration of 0.2mg/mL, the culture was continued until OD reached 0.6, IPTG was added to the solution to a final concentration of 0.1mM, and the solution was fermented at 25 ℃ for 18 hours, and then cells were collected by centrifugation and disrupted. The cells were disrupted for 15min and inclusion bodies were collected. The molecular weight of the target protein is 35.2 KDa. The expression results are shown in FIG. 7. The overall solubility expression of the target protein is less.
From the results of examples 4-7, it is clear that soluble expression of ketoreductase could be improved by not any chaperone vector.
Example 8
Ketoreductase- -catalytic reaction of crude enzyme liquid of pGro7 recombinant bacteria:
(1)100mg of substrate (ethyl 4-chloroacetoacetate), 4.4g of isopropanol are dissolved and mixed.
(2)20mg of the crude enzyme solution of the ketoreductase- -pGro7 disrupted cell of example 3 (1) and 20mg of NADP were put in 2.5mL of 200mM phosphate buffer and incubated at 25 ℃.
(3) Adding the substrate mixed solution into the enzyme solution mixed solution, reacting for 2 hours at 25 ℃, sampling, and analyzing by gas chromatography. The conversion was 100% at 2 hours, as determined by gas chromatography. As shown in fig. 8, the peak at t ═ 7.687 is ethyl 4-chloro-3-hydroxy-butyrate, the target compound.
In summary, compared with the prior art, the invention has the following advantages:
1. the invention discovers for the first time that the soluble expression of the ketoreductase can be realized by combining the ketoreductase with the pGro7 vector, and an effective soluble expression scheme aiming at the ketoreductase is obtained;
2. by inducing recombinant bacteria comprising molecular chaperone vectors and expressing ketoreductase at lower temperatures, substantial improvement in soluble expression of ketoreductase can be achieved, and the formation of inclusion bodies is significantly reduced while full enzymatic activity is retained.
The above are only examples of the present application, and do not limit the scope of the present application. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. All embodiments need not be described or illustrated herein. The technical solutions like this formed by equivalent transformation or equivalent substitution fall within the protection scope of the present application.
Sequence listing
<110> Shanghai Hequan drug development Co Ltd
SHANGHAI SYNTHEALL PHARMACEUTICALS Co.,Ltd.
<120> recombinant bacterium and method for expressing ketoreductase
<130> 218335 1CNCN
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1041
<212> DNA
<213> Rhodococcus ruber (Rhodococcus ruber)
<400> 1
atgaaagccc tccagtacac cgagatcggc tccgagccgg tcgtcgtcga cgtccccacc 60
ccggcgcccg ggccgggtga gatcctgctg aaggtcaccg cggccggctt gtgccactcg 120
gacatcttcg tgatggacat gccggcagag cagtacatct acggtcttcc cctcaccctc 180
ggccacgagg gcgtcggcac cgtcgccgaa ctcggcgccg gcgtcaccgg attcgagacg 240
ggggacgccg tcgccgtgta cgggccgtgg gggtgcggtg cgtgccacgc gtgcgcgcgc 300
ggccgggaga actactgcac ccgcgccgcc gagctgggca tcaccccgcc cggtctcggc 360
tcgcccgggt cgatggccga gtacatgatc gtcgactcgg cgcgccacct cgtcccgatc 420
ggggacctcg accccgtcgc ggcggttccg ctcaccgacg cgggcctgac gccgtaccac 480
gcgatctcgc gggtcctgcc cctgctggga cccggctcga ccgcggtcgt catcggggtc 540
ggcggactcg ggcacgtcgg catccagatc ctgcgcgccg tcagcgcggc ccgcgtgatc 600
gccgtcgatc tcgacgacga ccgactcgcg ctcgcccgcg aggtcggcgc cgacgcggcg 660
gtgaagtcgg gcgccggggc ggcggacgcg atccgggagc tgaccggcgg tgagggcgcg 720
acggcggtgt tcgacttcgt cggcgcccag tcgacgatcg acacggcgca gcaggtggtc 780
gcgatcgacg ggcacatctc ggtggtcggc atccatgccg gcgcccacgc caaggtcggc 840
ttcttcatga tcccgttcgg cgcgtccgtc gtgacgccgt actggggcac gcggtccgag 900
ctgatggacg tcgtggacct ggcccgtgcc ggccggctcg acatccacac cgagacgttc 960
accctcgacg agggacccac ggcctaccgg cggctacgcg agggcagcat ccgcggccgc 1020
ggggtggtcg tcccgggctg a 1041
<210> 2
<211> 346
<212> PRT
<213> Rhodococcus ruber (Rhodococcus ruber)
<400> 2
Met Lys Ala Leu Gln Tyr Thr Glu Ile Gly Ser Glu Pro Val Val Val
1 5 10 15
Asp Val Pro Thr Pro Ala Pro Gly Pro Gly Glu Ile Leu Leu Lys Val
20 25 30
Thr Ala Ala Gly Leu Cys His Ser Asp Ile Phe Val Met Asp Met Pro
35 40 45
Ala Glu Gln Tyr Ile Tyr Gly Leu Pro Leu Thr Leu Gly His Glu Gly
50 55 60
Val Gly Thr Val Ala Glu Leu Gly Ala Gly Val Thr Gly Phe Glu Thr
65 70 75 80
Gly Asp Ala Val Ala Val Tyr Gly Pro Trp Gly Cys Gly Ala Cys His
85 90 95
Ala Cys Ala Arg Gly Arg Glu Asn Tyr Cys Thr Arg Ala Ala Glu Leu
100 105 110
Gly Ile Thr Pro Pro Gly Leu Gly Ser Pro Gly Ser Met Ala Glu Tyr
115 120 125
Met Ile Val Asp Ser Ala Arg His Leu Val Pro Ile Gly Asp Leu Asp
130 135 140
Pro Val Ala Ala Val Pro Leu Thr Asp Ala Gly Leu Thr Pro Tyr His
145 150 155 160
Ala Ile Ser Arg Val Leu Pro Leu Leu Gly Pro Gly Ser Thr Ala Val
165 170 175
Val Ile Gly Val Gly Gly Leu Gly His Val Gly Ile Gln Ile Leu Arg
180 185 190
Ala Val Ser Ala Ala Arg Val Ile Ala Val Asp Leu Asp Asp Asp Arg
195 200 205
Leu Ala Leu Ala Arg Glu Val Gly Ala Asp Ala Ala Val Lys Ser Gly
210 215 220
Ala Gly Ala Ala Asp Ala Ile Arg Glu Leu Thr Gly Gly Glu Gly Ala
225 230 235 240
Thr Ala Val Phe Asp Phe Val Gly Ala Gln Ser Thr Ile Asp Thr Ala
245 250 255
Gln Gln Val Val Ala Ile Asp Gly His Ile Ser Val Val Gly Ile His
260 265 270
Ala Gly Ala His Ala Lys Val Gly Phe Phe Met Ile Pro Phe Gly Ala
275 280 285
Ser Val Val Thr Pro Tyr Trp Gly Thr Arg Ser Glu Leu Met Asp Val
290 295 300
Val Asp Leu Ala Arg Ala Gly Arg Leu Asp Ile His Thr Glu Thr Phe
305 310 315 320
Thr Leu Asp Glu Gly Pro Thr Ala Tyr Arg Arg Leu Arg Glu Gly Ser
325 330 335
Ile Arg Gly Arg Gly Val Val Val Pro Gly
340 345

Claims (10)

1. A recombinant bacterium comprising a chaperone vector including a pGro7 vector and expressing a ketoreductase.
2. The recombinant bacterium of claim 1, wherein the ketoreductase is derived from Rhodococcus ruber strain SD 3.
3. The recombinant bacterium of claim 1, wherein the ketoreductase has an amino acid sequence as set forth in SEQ ID NO: 2, respectively.
4. The recombinant bacterium of claim 1, wherein the recombinant bacterium comprises escherichia coli, preferably wherein the escherichia coli comprises escherichia coli BL21(DE) 3.
5. A method of expressing a ketoreductase enzyme, the method comprising:
a. providing a recombinant bacterium according to any one of claims 1-4;
b. culturing the recombinant strain; and
c. inducing the recombinant bacteria at a lower temperature to express the ketoreductase.
6. The method of claim 1, wherein the lower temperature is 10-35 ℃, preferably 15-30 ℃, more preferably 20-25 ℃.
7. The method of claim 1, wherein the induction lasts for 5 to 50 hours, preferably 10 to 20 hours, more preferably 12 to 16 hours.
8. The method of claim 1, wherein said inducing comprises the use of an inducing agent, preferably wherein said inducing agent comprises one or more of: arabinose and IPTG.
9. The method of claim 8, wherein the inducing comprises:
(1) adding arabinose when OD is cultured to 0.2-0.4, preferably 0.25-0.35, more preferably 0.3; and
(2) when the culture OD reached 0.5 to 0.7, preferably 0.55 to 0.65, more preferably 0.6, IPTG was added.
10. The method of claim 9, wherein (1) comprises adding 0.1-0.5mg/mL, 0.15-0.3mg/mL, more preferably 0.2mg/mL of arabinose, and (2) comprises adding 0.05-0.3mmol/L, preferably 0.08-0.15mmol/L, more preferably 0.1mmol/L of IPTG.
CN202111649747.6A 2021-12-30 2021-12-30 Recombinant bacterium and method for expressing ketoreductase Pending CN114395519A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103923957A (en) * 2013-05-10 2014-07-16 苏州汉酶生物技术有限公司 Chirality N-protective pipradrol biologically-preparing method
CN108865962A (en) * 2018-07-09 2018-11-23 南京林业大学 It is a kind of can solution expression with high efficiency 4- alpha-glycosyl transferase colibacillus engineering
CN108998462A (en) * 2018-08-01 2018-12-14 武汉康复得生物科技股份有限公司 The escherichia expression system and its application method of the recombinant protein containing manganese ion
CN109486782A (en) * 2018-11-26 2019-03-19 江南大学 A kind of method that molecular chaperones coexpression improves sucrose phosphorylase expression efficiency
CN111057686A (en) * 2019-12-23 2020-04-24 浙江大学 Alcohol dehydrogenase mutant and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103923957A (en) * 2013-05-10 2014-07-16 苏州汉酶生物技术有限公司 Chirality N-protective pipradrol biologically-preparing method
CN108865962A (en) * 2018-07-09 2018-11-23 南京林业大学 It is a kind of can solution expression with high efficiency 4- alpha-glycosyl transferase colibacillus engineering
CN108998462A (en) * 2018-08-01 2018-12-14 武汉康复得生物科技股份有限公司 The escherichia expression system and its application method of the recombinant protein containing manganese ion
CN109486782A (en) * 2018-11-26 2019-03-19 江南大学 A kind of method that molecular chaperones coexpression improves sucrose phosphorylase expression efficiency
CN111057686A (en) * 2019-12-23 2020-04-24 浙江大学 Alcohol dehydrogenase mutant and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Rhodococcus ruber padh gene (partial), sadh gene and sudh gene" *
朱玉贤 等: "现代分子生物学", 高等教育出版社, pages: 353 *
邓通 等: "基于分子伴侣策略提高NADPH依赖型醇脱氢酶的异源可溶性表达", vol. 40, no. 8, pages 24 - 32 *

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