CN113025601B

CN113025601B - Optimized expression and application of nitrilase promoter

Info

Publication number: CN113025601B
Application number: CN201911355889.4A
Authority: CN
Inventors: 阮礼涛; 钟胡军; 丁扬阳; 陈茜; 顾虹
Original assignee: Shanghai Aobo Biomedical Co ltd
Current assignee: Shanghai Aobo Biomedical Co ltd
Filing date: 2019-12-25
Publication date: 2024-06-21
Anticipated expiration: 2039-12-25

Abstract

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

Description

Optimized expression and application of nitrilase promoter

Technical Field

The invention relates to the screening, replacement and application of a promoter of an escherichia coli expression system, in particular to the optimization of a recombinant expression system of recombinant nitrilase, and the application of an enzyme or recombinant cells prepared by the technology in the preparation of gabapentin intermediate 1-cyanocyclohexyl acetic acid.

Background

Gabapentin, chemical name 1- (aminomethyl) -cyclohexylacetic acid, was first marketed in the uk in 1993 by the company Warner-Lambert. Is generally used for treating epilepsy and is also the first choice for treating neuropathic pain such as diabetic neuritis, post-herpetic severe pain, central nervous pain and the like. As early as 2005, the sales of gabapentin reached $ 30 billion, accounting for 1/3 of the sales revenue of antiepileptic drugs. According to WTO statistics, about 5000 thousands of epileptic patients exist worldwide, the lifelong prevalence of Chinese epileptic patients is over 800 ten thousand, the annual new incidence is about 40 ten thousand, and the demand for gabapentin bulk drugs is continuously increased in the global scope. Currently, gabapentin compound patents have expired.

The chemical process route is mainly referred to in patent EP0414262A2, in STEP3 and STEP4, HCl (0.97 ton), 3M NaOH solution (2.82M ³), ethanol (0.71 ton), toluene (3.93M ³), methanol (0.47M ³) are consumed per 1 ton of product produced, the overall yield of the two STEPs being 78% and with the formation of 1- (carboxymethyl) -cyclohexanecarboxylic acid impurities. The route is as follows:

In the earlier stage work, the nitrilase gene nit is cloned from the genome of Acidovorax facilis (Acidovorax facilis) ATCC11228 to construct E.coli BL21 (DE 3)/pET 28a-T7-nit (constructed by utilizing commercial pET28a plasmid, which is marked as pET28a-T7-nit for distinguishing a promoter), recombinant bacteria are constructed, the STEP3 and STEP4 are replaced by an enzymatic method, the single-STEP yield is more than 95%, and the catalytic reaction can be completed under normal temperature without using strong acid, strong alkali and organic reagent, so that the method has remarkable advantages compared with the prior art. The route is as follows:

The pET plasmid selected by the recombinant expression system is the 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 rate of synthesizing mRNA by the high-activity T7 RNA polymerase is 5 times faster than that by the E.coli RNA polymerase, when the two are simultaneously present, the transcription of host background genes cannot compete with a T7 expression system, and almost all cell resources are used for expressing target proteins; after induction expression, the recombinant protein can reach more than 50% of the total cell protein in a few hours. However, there is often a problem in pET expression systems that the expression yield is too high, the protein synthesis rate is too fast, and there is insufficient time for correct folding, resulting in inclusion body precipitation after induction, affecting cell growth and functional protein expression. The prevention of inclusion bodies in the study mainly adopts a mode of reducing the induction temperature and the induction dosage, but in many cases, optimizing the induction conditions cannot alleviate the need of regulating the expression intensity from the transcription and translation level of the inclusion body production (Ruan LT,Zheng RC and Zheng YG.Journal of Industrial Microbiology&Biotechnology,2016,43,1071-1083),.

The E.coli BL21 (DE 3)/pET 28a-T7-nit constructed by the research has serious inclusion body problems, and the optimized induction expression condition can not be relieved, so that the problems of serious inhibition of bacterial growth, unstable fermentation process, high biocatalyst cost and the like are caused. Therefore, the promoter replacement technology is utilized to regulate the transcription intensity, remove the growth bottleneck of thalli, have great significance for further advancing the production process of the gabapentin enzymatic method, and simultaneously provide a new scheme for improving the soluble expression of recombinant proteins.

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 of thalli and the stability of fermentation process, and provide a more efficient biocatalyst for the chemical enzyme method process of gabapentin.

The technical scheme adopted by the invention is as follows:

The nitrilase NIT is obtained by amplifying the genome of Acidovorax facilis (Acidovorax facilis) ATCC11228 by a gene cloning method, and a recombinant escherichia coli cell is constructed, and the nitrilase activity of preparing 1-cyanocyclohexyl acetic acid by selectively hydrolyzing 1-cyanocyclohexyl acetonitrile is verified, wherein the reaction formula is as follows:

the coding gene of A.fasciolis ATCC11228 NIT is obtained by cloning. The nucleotide sequence of the gene is shown in SEQ ID NO: 1. The amino acid sequence of nitrilase expressed by nit gene is shown in SEQ ID NO: 2.

The invention also relates to a recombinant vector containing the coding gene and recombinant genetic engineering bacteria obtained by utilizing the recombinant vector to transform. The recombinant vector is constructed by connecting the nucleotide sequence of the NIT encoding gene of the invention to various vectors by a conventional method. The vector may be any of a variety of vectors conventional in the art, such as various plasmids, phage or viral vectors, and the like, preferably pET28a.

The invention also provides a genetically engineered bacterium containing the coding gene or the recombinant vector. The genetically engineered bacterium can be obtained by transforming the recombinant expression vector of the invention into a host microorganism. The host microorganism may be any of various host microorganisms conventional in the art as long as it is satisfied that the recombinant expression vector can stably self-replicate and that the nit gene of the present invention carried can be expressed efficiently. The invention selects escherichia coli, preferably escherichia coli E.coli BL21 (DE 3). And transforming the recombinant plasmid pET28a-T7-nit into E.coli BL21 (DE 3) to obtain recombinant E.coli BL21 (DE 3)/pET 28a-T7-nit.

In particular, the invention carries out promoter screening and transformation on pET series commercial plasmids, adjusts the transcription rate of nit genes, solves the problem of inclusion bodies in the recombinant protein expression process, removes the growth bottleneck of engineering bacteria, and improves the activity of the bacteria by more than 2 times. The application is as follows: recombinant expression plasmids pET28a-T7-nit constructed by commercial plasmids are used as templates, expression plasmids such as pET28a-trc-nit, pET28a-trp-nit, pET28a-tac-nit, pET28a-T5-nit and the like are constructed by using a promoter replacement technology, and are converted into E.coli BL21 (DE 3) to construct an efficient biocatalyst, and 1-cyanocyclohexyl acetonitrile is catalyzed in water or buffer solution to prepare the 1-cyanocyclohexyl acetic acid.

The beneficial effects of the invention are mainly as follows: the promoter screening and replacement scheme is provided, commercial plasmids are modified, the expression intensity of recombinant proteins is regulated, the inclusion body effect is removed, and a new solution is provided for the inclusion body problem caused by the excessively high expression rate; the fermentation enzyme activity of the promoter mutant strain constructed by the invention is improved by more than 5.4 times compared with that of the original E.coliBL21 (DE 3)/pET 28a-T7-nit, so that the thallus fermentation cost and the biocatalyst consumption are greatly reduced; the 1-cyanocyclohexyl acetic acid of the gabapentin intermediate is prepared by the mutant strain of the promoter, 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 alkali and organic reagents, and the like, and has great industrialized application prospect.

Drawings

FIG. 1 is a map of the expression vector pET28a-T7-nit and promoter replacement;

FIG. 2 is a SDS-PAGE pattern of a sample of the disrupted supernatant of the induced expression of the engineering bacteria; lane 1 is protein molecular weight Marker, lane 2 is E.coli BL21 (DE 3)/pET 28a-T7-nit, lane 3 is E.coli BL21 (DE 3)/pET 28a-T5-nit, lane 4 is E.coli BL21 (DE 3)/pET 28a-tac-nit, lane 4 is E.coli BL21 (DE 3)/pET 28a-trc-nit, lane 5 is E.coli BL21 (DE 3)/pET 28a-trp-nit;

FIG. 3 is a SDS-PAGE map of an engineering bacterium induced expression disruption sediment sample; lane 1 is protein molecular weight Marker, lane 2 is E.coli BL21 (DE 3)/pET 28a-T7-nit, lane 3 is E.coli BL21 (DE 3)/pET 28a-T5-nit, lane 4 is E.coli BL21 (DE 3)/pET 28a-tac-nit, lane 4 is E.coli BL21 (DE 3)/pET 28a-trc-nit, lane 5 is E.coli BL21 (DE 3)/pET 28a-trp-nit.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

LB liquid medium: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride and natural pH.

Fermentation medium: peptone 16g/L, yeast 10g/L, sodium chloride 5g/L, pH 7.0.+ -. 0.1.

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 nitrilase is obtained by a gene cloning method, A.fasalis ATCC11228 genome is extracted by adopting a bacterial genome extraction kit (Takara) as a template, and a primer pair nit gene is designed for PCR amplification according to NCBI database A.fasalis nitrilase homology analysis, wherein the primer sequence is shown in SEQ ID NO:3 and SEQ ID NO:4. the PCR system (100. Mu.L) was: template (10 ng/. Mu.L) 0.5. Mu.L, upstream primer (50. Mu.M) 0.5. Mu.L, downstream primer (50. Mu.M) 0.5. Mu.L, dNTP mix (2.5 mM) 8. Mu.L, 5X PRIMESTAR BUFFER. Mu.L, ddH ₂ O69.5. 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, and 30 times (2) - (4) steps and (5) extension at 72℃for 5min.

And (3) taking a PCR product, performing glue recovery to obtain a target gene, performing double enzyme digestion and recovery treatment on the target gene by using NcoI and XhoI restriction enzymes, and connecting the fragment with a commercial vector pET28a treated by the same restriction enzymes by using T4 DNA ligase at 16 ℃ overnight to construct a recombinant expression vector pET28a-nit. Transforming the constructed recombinant expression vector pET28a-nit into E.coli BL21 (DE 3) competent cells, coating the competent cells on LB plates containing 50 mug/mL kanamycin at a final concentration, and culturing overnight at 37 ℃; colony PCR identification is carried out by randomly picking clones from colonies growing on a flat plate, and positive clone sequencing verification shows that the recombinant expression vector pET28a-nit is successfully transformed into an expression host E.coli BL21 (DE 3), and the nit gene is successfully cloned to 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

Culturing recombinant bacteria E.coli BL21 (DE 3)/pET 28a-nit constructed in example 1, extracting plasmids as a promoter mutation template, designing primers for mutating the recombinant plasmids, wherein the sequences of the primers are shown in SEQ ID NO: 5-SEQ ID NO:12. the recombinant expression plasmid pET28a-T7-nit map and the promoter replacement scheme are shown in figure 1. The PCR system (100. Mu.L) was: template (10 ng/. Mu.L) 0.5. Mu.L, upstream primer (50. Mu.M) 0.5. Mu.L, downstream primer (50. Mu.M) 0.5. Mu.L, dNTP mix (2.5 mM) 8. Mu.L, 5X PRIMESTAR BUFFER. Mu.L, ddH ₂ O69.5. 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, and 30 times (2) - (4) steps and (5) extension at 72℃for 5min.

Taking PCR products to perform DpnI treatment on digestion templates, converting the 4 groups of digestion products into E.coli BL21 (DE 3) competent cells, coating the competent cells on LB plates containing 50 mug/mL kanamycin at a final concentration, and culturing overnight at 37 ℃; colony PCR identification is carried out by randomly picking clones from colonies growing on a flat plate, and 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

Inoculating the genetically engineered bacteria pET28a-NIT constructed in the examples 1 and 2 into a fermentation culture medium containing 50 mug/mL kanamycin, culturing at 37 ℃ until the cell concentration OD ₆₀₀ is 0.6-0.8, adding IPTG with the final concentration of 0.1mmol/L into the fermentation culture medium, performing induction culture at 28 ℃ overnight, centrifuging the culture solution at 4 ℃ at 12000rpm for 5min, discarding the supernatant, and collecting NIT wet cells expressed by different promoters. 1g of wet thalli are weighed and suspended in 10mL of phosphate buffer solution (pH 7.0), ultrasonic crushing is carried out, analysis is carried out on thalli crushing supernatant and precipitate by utilizing SDS-PAGE protein denaturation electrophoresis (figures 2 and 3), and the result shows that the expression quantity of pET28a-T7-nit recombinant protein is maximum, but the expression rate is too fast, the inclusion body proportion is the highest, and only a trace amount of inclusion bodies are generated in recombinant engineering bacteria replaced by the promoter; the concentration of pET28a-T5-nit soluble protein is significantly higher than that of the original commercial plasmid.

Example 4: determination of NIT bacterial body amounts and enzyme activities of different promoters

NIT wet cells expressed by the different promoters in example 3 were collected by centrifugation at 12000rpm for 5min at 4℃to determine cell concentration, and the obtained cells were used to catalyze the substrate 1-cyanocyclohexylacetonitrile.

The catalytic system comprises the following components and catalytic conditions: to 10mL of the phosphate buffer (50 mmol/L, pH 7.0) was added wet cells (final concentration 5.0 g/L), and 1-cyanocyclohexylacetonitrile (final concentration 60.0 g/L) to construct a reaction system. The specific enzyme activity of the thalli is sampled and detected after the reaction is carried out for 30min at the reaction temperature of 35 ℃ and the rotating speed of 200rpm, and the enzyme activity of the unit fermentation broth is calculated.

The measurement results are shown in Table 1, the cell quantity of all 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% and 264.6%, and the specific enzyme activities of corresponding fermentation liquor are improved to 491.5% and 646.2%.

TABLE 1 comparison of the cell concentrations and enzyme activities of different promoters

	T7	trc	trp	tac	T5
						Concentration of thallus (g/L)	4.3	11.6	11.0	12.0	10.5
Specific enzyme activity (U/g _{Wet cell})	156.6	12.5	0.0	275.8	414.4
						Specific enzyme activity (U/L) of fermentation broth	673.4	145.0	0.0	3309.6	4351.2

Example 5: preparation of 1-cyanocyclohexyl acetic acid by recombinant bacterium pET28a-T7-nit catalysis

PET28a-T7-nit cells obtained in the method of example 3 are used as a catalyst, and 1-cyano-cyclohexyl acetonitrile is used as a substrate.

The catalytic system comprises the following components and reaction conditions: 50mL of phosphate buffer (50 mmol/L, pH 7.0) is added with recombinant pET28a-T7-nit wet thalli (final concentration 20 g/L), 1-cyano-cyclohexyl acetonitrile (final concentration 100 g/L) forms a reaction system, the rotation speed is 200rpm at 35 ℃, the thalli are removed by centrifugation for 18h, and the reaction yield of the product 1-cyano-cyclohexyl acetic acid detected by HPLC is more than 94.9%.

Example 6: preparation of 1-cyanocyclohexyl acetic acid by recombinant bacterium pET28a-T7-nit catalysis

The procedure of example 5 was repeated except that the amount of pET28a-T7-nit wet cells 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 1-cyanocyclohexylacetic acid was 95.3% by HPLC.

Example 7: preparation of 1-cyanocyclohexyl acetic acid by recombinant bacterium pET28a-trc-nit catalysis

The recombinant bacterium used in example 5 was changed to pET28a-trc-nit, the amount of the added bacterium was 20g/L, the other operations were the same as those in example 5, the bacterium was removed by centrifugation after 18 hours of reaction, and the reaction yield of the product 1-cyanocyclohexylacetic acid was 17.5% by HPLC.

Example 8: preparation of 1-cyanocyclohexyl acetic acid by recombinant bacterium pET28a-tac-nit catalysis

The recombinant bacterium used in example 5 was changed to pET28a-tac-nit, the added amount of the bacterium was 20g/L, the other operations were the same as those in example 5, the bacterium was removed after 18 hours of reaction, and the reaction yield of the product 1-cyanocyclohexylacetic acid was 96.3% by HPLC.

Example 9: preparation of 1-cyanocyclohexyl acetic acid by recombinant bacterium pET28a-T5-nit catalysis

PET28a-T5-nit cells obtained in the method of example 3 are used as a catalyst, and 1-cyano-cyclohexyl acetonitrile is used as a substrate.

The catalytic system comprises the following components and reaction conditions: 500mL of phosphate buffer (50 mmol/L, pH 7.0) is added with recombinant pET28a-T5-nit wet thalli (final concentration 20 g/L), 1-cyano-cyclohexyl acetonitrile (final concentration 100 g/L) forms a reaction system, the rotation speed is 200rpm at 35 ℃, the thalli are removed by centrifugation for 18h, and the reaction yield of the product 1-cyano-cyclohexyl acetic acid detected by HPLC is more than 99.5%.

Sequence listing

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Claims

1. A method for constructing a nitrilase high-efficiency expression system, which is characterized by comprising the following steps: carrying out promoter replacement on a recombinant nitrilase expression plasmid constructed in a commercialized vector pET-T7-nit, wherein the replaced promoter is selected from T5 promoters; wherein, the nucleotide sequence of nit gene is shown as SEQ ID NO:1, the amino acid sequence of the nitrilase expressed by nit gene is shown as SEQ ID NO: 2.

2. Use of a method for constructing a high-efficiency expression system of a nitrilase according to claim 1 for biocatalytically preparing gabapentin intermediates.

3. The application of claim 2, wherein the application is: the nitrilase high-efficiency expression system as defined in claim 1 is used as a catalyst to catalyze the regioselective hydrolysis reaction of 1-cyanocyclohexyl acetonitrile in water or a buffer system to prepare the 1-cyanocyclohexyl acetic acid, wherein the reaction concentration of the substrate 1-cyanocyclohexyl acetonitrile is 10-250g/L.