CN113025601A - Nitrilase promoter optimized expression and application - Google Patents

Nitrilase promoter optimized expression and application Download PDF

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CN113025601A
CN113025601A CN201911355889.4A CN201911355889A CN113025601A CN 113025601 A CN113025601 A CN 113025601A CN 201911355889 A CN201911355889 A CN 201911355889A CN 113025601 A CN113025601 A CN 113025601A
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recombinant
nit
nitrilase
expression
promoter
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阮礼涛
钟胡军
丁扬阳
陈茜
顾虹
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Shanghai Aobo Bio Pharmaceutical Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/002—Nitriles (-CN)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)

Abstract

The invention discloses a pET commercial plasmid promoter screening and replacing method. The method improves the correct folding proportion of the nitrilase, increases the soluble expression and removes the inhibition of the accumulation of inclusion bodies on the growth of thalli by regulating the heterologous expression of the nitrilase from the transcription level; the shake flask induction expression is carried out on the promoter mutant strain, and the concentration of thalli, the specific enzyme activity and the specific enzyme activity of the fermentation solution are respectively improved by 1.4 times, 1.6 times and 5.5 times compared with the commercial plasmid; simultaneously in the reaction for preparing gabapentin intermediate 1-cyanocyclohexylacetic acid in a catalytic manner; the catalytic efficiency of the promoter mutant strain is improved by more than 100 percent compared with that of the original strain.

Description

Nitrilase promoter optimized expression and application
Technical Field
The invention relates to screening, replacement and application of a promoter of an escherichia coli expression system, in particular to optimization of a recombinant nitrilase recombinant expression system and application of enzyme or recombinant cells prepared by the technology in preparation of gabapentin intermediate 1-cyanocyclohexylacetic acid.
Background
Gabapentin, chemically known as 1- (aminomethyl) -cyclohexylacetic acid, was first marketed in 1993 in the uk by the company Warner-Lambert, formerly. Is generally used for treating epilepsy and is the first choice for treating neuropathic pains such as diabetic neuritis, acute pain after herpes, central neuropathic pain and the like. As early as 2005, gabapentin sold $ 30 billion, which accounts for 1/3 in the revenue from the sale of antiepileptic drugs. According to WTO statistics, about 5000 million epileptic patients exist in the world, more than 800 million Chinese epileptic patients have lifetime prevalence rate of 0.7%, 40 new cases of each year are about, and the global demand for gabapentin raw material medicine is continuously increased. At present, gabapentin compound patents have expired.
Chemical Process route reference is made mainly to patent EP0414262A2, in STEP3 and STEP4 STEPs, HCl (0.97 tons), 3M NaOH solution (2.82M) is consumed for each 1 ton of product produced3) Ethanol (0.71 ton), toluene (3.93 m)3) Methanol (0.47 m)3) The overall yield of the two-step reaction is 78% with the formation of 1- (carboxymethyl) -cyclohexanecarboxylic acid as an impurity. The route is as follows:
Figure BDA0002335916460000011
in the early work, a nitrilase gene nit is cloned from an Acidovorax fastigii (Acidovorax fascilis) ATCC11228 genome, an E.coli BL21(DE3)/pET28a-T7-nit (constructed by utilizing a commercial pET28a plasmid and marked as pET28a-T7-nit for facilitating the distinction of promoters) recombinant bacterium is constructed, the STEP3 and STEP4 are replaced by utilizing an enzyme method, the single-STEP yield is more than 95%, strong acid and strong base and organic reagents are not needed, the catalytic reaction can be completed at normal temperature, and the method has obvious advantages compared with the original process. The route is as follows:
Figure BDA0002335916460000012
the pET plasmid selected by the recombinant expression system is a prokaryotic expression vector which is most widely applied in scientific research and industrial production. The pET series plasmid integrates a T7 promoter, the promoter is exclusively controlled by T7 RNA polymerase, the mRNA synthesis rate of the high-activity T7 RNA polymerase is 5 times faster than that of the Escherichia coli RNA polymerase, when the two exist at the same time, the transcription of the host background gene can not compete with the T7 expression system, and almost all cell resources are used for expressing the target protein; after induction expression, the recombinant protein can reach over 50 percent of total cell protein within a few hours. However, the pET expression system often has the problems of high expression yield, high protein synthesis rate and insufficient time for correct folding, so that inclusion body precipitation is formed after induction, and the thallus growth and the protein function expression are influenced. Prevention of inclusion bodies was mainly done by lowering the induction temperature and the induction dose, but in many cases, optimization of induction conditions did not alleviate inclusion body production (Ruan LT, Zheng RC and Zheng YG. journal of Industrial Microbiology & Biotechnology,2016,43,1071-1083), requiring regulation of expression intensity from both transcriptional and translational levels.
The E.coli BL21(DE3)/pET28a-T7-nit constructed in the research has a serious inclusion body problem, and the optimized induced expression condition can not be relieved, so that the problems of serious thallus growth inhibition, unstable fermentation process, high biocatalyst cost and the like are caused. Therefore, the promoter replacement technology is used for regulating and controlling the transcription strength and removing the bottleneck of thallus growth, has very important significance for further promoting the production process of the gabapentin enzyme method, and simultaneously provides a new scheme for improving the soluble expression of the recombinant protein.
Disclosure of Invention
The invention aims to provide a new scheme for increasing the soluble expression of nitrilase, remove the growth inhibition of inclusion body accumulation on thalli, improve the specific enzyme activity and the fermentation process stability of the thalli, and provide a more efficient biocatalyst for a gabapentin chemical enzyme process.
The technical scheme adopted by the invention is as follows:
the nitrilase NIT is obtained by amplifying Acidovorax fascilis ATCC11228 genome through a gene cloning method, a recombinant escherichia coli cell is constructed, and the nitrilase NIT is verified to have hydrolase activity for selectively hydrolyzing 1-cyanocyclohexylacetonitrile to prepare 1-cyanocyclohexylacetic acid, and the reaction formula is as follows:
Figure BDA0002335916460000021
the coding gene of A.facilis ATCC11228 NIT is obtained by cloning. The nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown. The amino acid sequence of the nitrilase expressed by nit gene is shown as SEQ ID NO: 2, respectively.
The invention also relates to a recombinant vector containing the coding gene and a recombinant gene engineering bacterium obtained by transforming the recombinant vector. The recombinant vector is constructed by connecting the nucleotide sequence of the NIT coding gene of the invention to various vectors by a conventional method. The vector may be any vector conventional in the art, such as any plasmid, phage or viral vector, and preferably pET28 a.
The invention also provides a genetic engineering bacterium containing the coding gene or the recombinant vector. The genetically engineered bacteria can be obtained by transforming the recombinant expression vector of the invention into host microorganisms. The host microorganism may be any of various host microorganisms conventionally used in the art, as long as it is satisfied that the recombinant expression vector can stably self-replicate and the carried nit gene of the present invention can be efficiently expressed. Coli BL21(DE3) is preferred. The recombinant plasmid pET28a-T7-nit is transformed into E.coli BL21(DE3) to obtain recombinant Escherichia coli E.coli BL21(DE3)/pET28 a-T7-nit.
Particularly, the invention screens and transforms a promoter of pET series commercial plasmids, regulates the transcription rate of nit genes, solves the problem of inclusion bodies in the expression process of recombinant proteins, removes the growth bottleneck of engineering bacteria, and simultaneously improves the activity of the bacteria by more than 2 times. The application is as follows: the recombinant expression plasmid pET28a-T7-nit constructed by commercial plasmid is used as a template, expression plasmids such as pET28a-trc-nit, pET28a-trp-nit, pET28a-tac-nit, pET28a-T5-nit and the like are constructed by utilizing a promoter replacement technology, and are converted into E.coli BL21(DE3) to construct a high-efficiency biocatalyst, and the 1-cyanocyclohexylacetonitrile is catalyzed in water or buffer solution to prepare the 1-cyanocyclohexylacetic acid.
The invention has the following beneficial effects: provides a promoter screening and replacing scheme, reforms commercial plasmid, regulates the expression intensity of recombinant protein, removes the effect of inclusion body, provides a new solution for the problem of inclusion body caused by too fast expression rate; compared with the original E.coliBL21(DE3)/pET28a-T7-nit, the fermentation enzyme activity of the promoter mutant strain constructed by the invention is improved by more than 5.4 times, so that the thallus fermentation cost and the dosage of a biocatalyst are greatly reduced; the gabapentin intermediate 1-cyanocyclohexylacetic acid is prepared by the promoter mutant strain, the substrate concentration can reach 100g/L, the reaction conversion rate is kept above 99%, and the method has the advantages of good selectivity, mild conditions, no need of using strong acid, strong base and organic reagent and the like, and has great industrial application prospect.
Drawings
FIG. 1 is a map of expression vector pET28a-T7-nit and promoter replacement;
FIG. 2 is an SDS-PAGE pattern of a supernatant sample of engineering bacteria induced expression; lane 1 is a protein molecular weight Marker, lane 2 is e.coli BL21(DE3)/pET28a-T7-nit, lane 3 is e.coli BL21(DE3)/pET28a-T5-nit, lane 4 is e.coli BL21(DE3)/pET28a-tac-nit, lane 4 is e.coli BL21(DE3)/pET28a-trc-nit, lane 5 is e.coli BL21(DE3)/pET28 a-trp-nit;
FIG. 3 is an SDS-PAGE pattern of an engineering bacteria induced expression fragmentation precipitation sample; lane 1 is a protein molecular weight Marker, lane 2 is e.coli BL21(DE3)/pET28a-T7-nit, lane 3 is e.coli BL21(DE3)/pET28a-T5-nit, lane 4 is e.coli BL21(DE3)/pET28a-tac-nit, lane 4 is e.coli BL21(DE3)/pET28a-trc-nit, and lane 5 is e.coli BL21(DE3)/pET28 a-trp-nit.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
LB liquid medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and natural pH.
Fermentation medium: 16g/L of peptone, 10g/L of yeast, 5g/L of sodium chloride and 7.0 +/-0.1 of pH.
Definition of enzyme activity unit (U): the amount of enzyme required to convert 1. mu. mol of 1-cyanocyclohexylacetonitrile in 1min at 35 ℃ and pH 7.0 was defined as 1U.
Example 1: construction of nitrilase gene engineering bacteria
The invention isThe nitrilase is obtained by a gene cloning method, a bacterial genome extraction kit (Takara) is adopted to extract A.faciis ATCC11228 genome as a template, a primer is designed to carry out PCR amplification on nit gene according to the homology analysis of the A.faciis nitrilase in NCBI database, and the sequence of the primer is shown in SEQ ID NO: 3 and SEQ ID NO: 4. the PCR system (100. mu.L) was: 0.5. mu.L template (10 ng/. mu.L), 0.5. mu.L upstream primer (50. mu.M), 0.5. mu.L downstream primer (50. mu.M), 8. mu.L dNTP mix (2.5mM), 5 XPrimeSTAR Buffer 20. mu.L ddH2O69.5. mu.L, PrimeSTAR DNA polymerase 1. mu.L. PCR procedure: (1) denaturation at 98 ℃ for 3min, (2) denaturation at 98 ℃ for 10s, (3) annealing at 60 ℃ for 5s, (4) extension at 72 ℃ for 1.2min, 30 times of the steps from (2) to (4) are circulated, and (5) extension at 72 ℃ for 5 min.
And carrying out gel recovery on the PCR product to obtain a target gene, carrying out double digestion and recovery treatment on the target gene by using restriction enzymes NcoI and XhoI, and connecting the fragment with a commercial vector pET28a treated by the same restriction enzyme at 16 ℃ overnight by using T4 DNA ligase to construct a recombinant expression vector pET28 a-nit. The constructed recombinant expression vector pET28a-nit was transformed into E.coli BL21(DE3) competent cells, spread on LB plate containing kanamycin to a final concentration of 50. mu.g/mL, and cultured overnight at 37 ℃; clones were randomly picked from colonies growing on the plate for colony PCR identification, positive clone sequencing verification, and the results showed that the recombinant expression vector pET28a-nit was successfully transformed into the expression host e.coli BL21(DE3), and the nit gene was successfully cloned into the NcoI and XhoI sites of pET-28 a. The nit gene consists of a 1110-nucleotide open reading frame and the expressed nitrilase consists of 369 amino acids.
Example 2: construction of recombinant strains containing different promoters
The recombinant bacterium E.coli BL21(DE3)/pET28a-nit constructed in the example 1 is cultured, plasmid is extracted to be used as a promoter mutation template, a primer is designed to carry out mutation on the recombinant plasmid, and the sequence of the primer is shown in SEQ ID NO: 5 to SEQ ID NO: 12. the map of the recombinant expression plasmid pET28a-T7-nit and the replacement scheme of the promoter are shown in figure 1. The PCR system (100. mu.L) was: 0.5. mu.L template (10 ng/. mu.L), 0.5. mu.L upstream primer (50. mu.M), 0.5. mu.L downstream primer (50. mu.M), 8. mu.L dNTP mix (2.5mM), 5 XPrimeSTAR Buffer 20. mu.LL，ddH2O69.5. mu.L, PrimeSTAR DNA polymerase 1. mu.L. PCR procedure: (1) denaturation at 98 ℃ for 3min, (2) denaturation at 98 ℃ for 10s, (3) annealing at 60 ℃ for 5s, (4) extension at 72 ℃ for 6.5min, 30 times of the steps (2) - (4) are circulated, and (5) extension at 72 ℃ for 5 min.
Taking PCR products to carry out DpnI treatment on a digestion template, transforming the 4 groups of digestion products into E.coli BL21(DE3) competent cells, coating the competent cells on an LB plate containing kanamycin with the final concentration of 50 mu g/mL, and culturing at 37 ℃ overnight; clones are randomly picked from colonies growing on the plate for colony PCR identification, positive clone sequencing verification shows that recombinant expression vectors pET28a-trc-nit, pET28a-trp-nit, pET28a-tac-nit and pET28a-T5-nit are successfully constructed and transformed into an expression host E.coli BL21(DE 3).
Example 3: preparation of NIT somatic cells with different promoters
The genetically engineered bacterium pET28a-nit constructed in examples 1 and 2 was inoculated into a fermentation medium containing 50. mu.g/mL kanamycin, and cultured at 37 ℃ to a cell density OD600The value is 0.6-0.8, then adding IPTG with the final concentration of 0.1mmol/L into the fermentation medium, inducing and culturing overnight at 28 ℃, centrifuging the culture solution at 4 ℃ and 12000rpm for 5min, removing the supernatant, and collecting NIT wet thalli expressed by different promoters. Weighing 1g of wet thallus, suspending the wet thallus in 10mL of phosphate buffer (pH 7.0), carrying out ultrasonic disruption, analyzing the disrupted supernatant and precipitate of the thallus by SDS-PAGE protein denaturing electrophoresis (figures 2 and 3), wherein the result shows that the expression amount of pET28a-T7-nit recombinant protein is maximum, but the expression rate is too high, the proportion of formed inclusion bodies is highest, and the recombinant engineering bacteria with promoter replaced only generates a trace amount of inclusion bodies; the concentration of pET28a-T5-nit soluble protein is obviously higher than that of the original commercial plasmid.
Example 4: NIT (nitrate) bacterium amount and enzyme activity determination of different promoters
NIT wet cells expressed by different promoters in example 3 were collected by centrifugation at 12000rpm for 5min at 4 ℃ and the cell concentration was measured to obtain a cell catalytic substrate, 1-cyanocyclohexylacetonitrile.
The composition of the catalytic system and the catalytic conditions are as follows: the reaction system was constituted by adding wet cells (final concentration: 5.0g/L) and 1-cyanocyclohexylacetonitrile (final concentration: 60.0g/L) to 10mL of a phosphate buffer (50mmol/L, pH 7.0). The reaction temperature is 35 ℃, the reaction is carried out for 30min under the condition of the rotating speed of 200rpm, the specific enzyme activity of the thalli is sampled and detected, and the enzyme activity of unit fermentation liquor is calculated.
The determination result is shown in table 1, the bacterial body quantity of all the promoter mutant strains is improved to more than 2 times compared with the original commercial plasmid expression strain, the specific enzyme activities of pET28a-T5-nit and pET28a-tac-nit are also improved to 176.1 percent and 264.6 percent of the original bacterial body, and the specific enzyme activities of corresponding fermentation liquids are improved to 491.5 percent and 646.2 percent.
TABLE 1 comparison of the concentrations of the different promoters and the enzyme activities
T7 trc trp tac T5
Cell concentration (g/L) 4.3 11.6 11.0 12.0 10.5
Specific enzyme activity (U/g)Wet thallus) 156.6 12.5 0.0 275.8 414.4
Specific activity of fermentation liquor (U/L) 673.4 145.0 0.0 3309.6 4351.2
Example 5: preparation of 1-cyanocyclohexylacetic acid by recombinant bacterium pET28a-T7-nit under catalysis
pET28a-T7-nit cell obtained in the method of example 3 was used as a catalyst, and 1-cyanocyclohexylacetonitrile was used as a substrate.
The composition of the catalytic system and the reaction conditions are as follows: 50mL of phosphate buffer solution (50mmol/L, pH 7.0) is added with recombinant pET28a-T7-nit wet thalli (final concentration is 20g/L), 1-cyanocyclohexylacetonitrile (final concentration is 100g/L) forms a reaction system, the rotating speed is 200rpm at 35 ℃, the thalli is removed by centrifugation after 18h of reaction, and the reaction yield of the product 1-cyanocyclohexylacetic acid is more than 94.9 percent by HPLC detection.
Example 6: preparation of 1-cyanocyclohexylacetic acid by recombinant bacterium pET28a-T7-nit under catalysis
The procedure of example 5 was otherwise the same except that the amount of wet pET28a-T7-nit cells added in example 5 was changed to 40g/L, and the cells were removed by centrifugation after 18 hours of reaction, and the reaction yield of the product, 1-cyanocyclohexylacetic acid, was 95.3% by HPLC.
Example 7: preparation of 1-cyanocyclohexylacetic acid by recombinant bacterium pET28a-trc-nit under catalysis
The recombinant strain used in example 5 was changed to pET28a-trc-nit, and the amount of added cells was 20g/L, the procedure was otherwise the same as in example 5, the cells were removed by centrifugation after 18 hours of reaction, and the reaction yield of the product 1-cyanocyclohexylacetic acid by HPLC was 17.5%.
Example 8: preparation of 1-cyanocyclohexylacetic acid by recombinant bacterium pET28a-tac-nit catalysis
The recombinant strain used in example 5 was changed to pET28a-tac-nit, the amount of added cells was 20g/L, the procedure was otherwise the same as in example 5, the cells were removed after 18 hours of reaction, and the reaction yield of the product 1-cyanocyclohexylacetic acid by HPLC was 96.3%.
Example 9: preparation of 1-cyanocyclohexylacetic acid by recombinant bacterium pET28a-T5-nit under catalysis
pET28a-T5-nit cell obtained in the method of example 3 was used as a catalyst, and 1-cyanocyclohexylacetonitrile was used as a substrate.
The composition of the catalytic system and the reaction conditions are as follows: 500mL phosphate buffer (50mmol/L, pH 7.0) is added with recombinant pET28a-T5-nit wet bacteria (final concentration 20g/L), 1-cyanocyclohexylacetonitrile (final concentration 100g/L) forms a reaction system, the temperature is 35 ℃, the rotation speed is 200rpm, the bacteria are removed by centrifugation after 18h of reaction, and the reaction yield of the product 1-cyanocyclohexylacetic acid is over 99.5 percent by HPLC detection.
Sequence listing
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<213> Artificial sequence ()
<400> 10
tacgagccga tgattaattg tcaacagctc atttcgcggg atcgagatct cg 52
<210> 11
<211> 56
<212> DNA
<213> Artificial sequence ()
<400> 11
tttatttgct ttgtgagcgg ataacaatta taatagggga attgtgagcg gataac 56
<210> 12
<211> 57
<212> DNA
<213> Artificial sequence ()
<400> 12
gttatccgct cacaaagcaa ataaattttt tatgaatttc gcgggatcga gatctcg 57

Claims (4)

1. The construction of a nitrilase high-efficiency expression system is characterized in that the method comprises the following steps: the plasmid for expressing recombinant nitrilase constructed from commercial vector pET-T7-nit was subjected to promoter selection and mutation replacement, and the selected replacement promoters include, but are not limited to, trc, trp, tac, T5 and lac series promoters.
2. Use of the method of claim 1 for removing the effects of inclusion bodies, wherein the use is: aiming at the problem of an inclusion body caused by an excessively high expression rate, a promoter with specific strength is selected for replacement, the gene transcription rate is regulated, the promoter replacement recombinant plasmid is transferred into a competent cell to construct recombinant engineering bacteria, after induction expression, the recombinant protein has sufficient time for correct folding, the inhibition of the inclusion body on the growth of the bacteria is removed, and the soluble expression of the recombinant nitrilase is enhanced.
3. Use of the nitrilase promoter replacement recombinant strain of claim 2 in biocatalytic production of gabapentin intermediates.
4. The use according to claim 3, characterized in that said use is: the nitrilase promoter replacement recombinant bacteria or corresponding immobilized recombinant bacteria are used as a catalyst to catalyze the regioselective hydrolysis reaction of 1-cyanocyclohexylacetonitrile in water or a buffer system to prepare the 1-cyanocyclohexylacetic acid, wherein the reaction concentration of the substrate 1-cyanocyclohexylacetonitrile is 10-250 g/L.
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