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Optimized expression of nitrilase promoter and application thereof

A nitrilase and promoter technology, applied in hydrolytic enzymes, enzymes, biochemical equipment and methods, etc., can solve the problems of inability to alleviate inclusion body production, high expression yield, inclusion body precipitation, etc., and achieve large-scale industrial application prospects , mild conditions, good selectivity

Pending Publication Date: 2021-06-25
SHANGHAI AOBO PHARMTECH INC LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, in the pET expression system, the expression yield is often too high, the protein synthesis rate is too fast, and there is not enough time for correct folding, resulting in the formation of inclusion body precipitation after induction, affecting bacterial growth and protein function expression
In the research, the main way to prevent inclusion bodies is to reduce the induction temperature and induction dose, but in many cases, optimizing the induction conditions cannot alleviate the inclusion body production (Ruan LT, Zheng RC and ZhengYG. Journal of Industrial Microbiology & Biotechnology, 2016, 43, 1071- 1083), it is necessary to regulate the expression intensity from the transcriptional and translational levels
[0008] The E.coli BL21(DE3) / pET28a-T7-nit constructed in this study has a serious inclusion body problem, which cannot be alleviated by optimizing the induced expression conditions, resulting in severe growth inhibition, unstable fermentation process, and high cost of biocatalysts.

Method used

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  • Optimized expression of nitrilase promoter and application thereof
  • Optimized expression of nitrilase promoter and application thereof
  • Optimized expression of nitrilase promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: construction of nitrilase genetically engineered bacteria

[0026] The nitrilase of the present invention is obtained by the method of gene cloning, adopts bacterial genome extraction kit (Takara) to extract A.facilis ATCC11228 genome as template, according to NCBI database A.facilis nitrilase homology analysis, design primer pair nit The gene is amplified by PCR, and the primer sequences are shown in SEQ ID NO:3 and SEQ ID NO:4. The PCR system (100 μL) is: template (10ng / μL) 0.5 μL, upstream primer (50 μM) 0.5 μL, downstream primer (50 μM) 0.5 μL, dNTPMixture (2.5mM) 8 μL, 5×PrimeSTAR Buffer 20 μL, ddH 2 O 69.5 μL, PrimeSTAR DNA polymerase 1 μL. PCR program: (1) Denaturation at 98°C for 3 minutes, (2) Denaturation at 98°C for 10 seconds, (3) Annealing at 60°C for 5 seconds, (4) Extension at 72°C for 1.2 minutes, cycle (2)-(4) steps 30 times, (5 ) at 72°C for 5 min.

[0027]Take the PCR product for gel recovery to obtain the target gene, use NcoI and Xh...

Embodiment 2

[0028] Example 2: Construction of recombinant strains containing different promoters

[0029] The recombinant strain E.coli BL21(DE3) / pET28a-nit constructed in Example 1 was cultivated, the plasmid was extracted as a promoter mutation template, and primers were designed to mutate the recombinant plasmid. See SEQ ID NO: 5 to SEQ ID NO: for the primer sequence 12. For the map of recombinant expression plasmid pET28a-T7-nit and the promoter replacement scheme, see figure 1 . The PCR system (100 μL) is: template (10ng / μL) 0.5 μL, upstream primer (50 μM) 0.5 μL, downstream primer (50 μM) 0.5 μL, dNTP Mixture (2.5mM) 8 μL, 5×PrimeSTAR Buffer 20 μL, ddH 2 O 69.5 μL, PrimeSTAR DNA polymerase 1 μL. PCR program: (1) Denaturation at 98°C for 3 minutes, (2) Denaturation at 98°C for 10 seconds, (3) Annealing at 60°C for 5 seconds, (4) Extension at 72°C for 6.5 minutes, cycle (2)-(4) steps 30 times, (5 ) at 72°C for 5 min.

[0030] Take the PCR product for DpnI treatment to digest the ...

Embodiment 3

[0031] Embodiment 3: the preparation of different promoter NIT bacterial cells

[0032] The genetically engineered bacteria pET28a-nit constructed in Examples 1 and 2 were inoculated into the fermentation medium containing 50 μg / mL kanamycin, and cultivated at 37°C to the cell concentration OD 600 If the value is 0.6-0.8, add a final concentration of 0.1mmol / L IPTG to the fermentation medium, induce culture overnight at 28°C, centrifuge the culture solution at 4°C, 12000rpm for 5min, discard the supernatant, and collect NIT expressed by different promoters wet bacteria. Each weighed 1g of wet bacteria, suspended in 10mL phosphate buffer (pH 7.0), ultrasonically disrupted, and analyzed the supernatant and precipitate of the broken bacteria by SDS-PAGE protein denaturation electrophoresis ( figure 2 and image 3 ), the results showed that the expression of pET28a-T7-nit recombinant protein was the largest, but because the expression rate was too fast, the proportion of inclus...

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Abstract

The invention discloses a method for screening and replacing a pET commercial plasmid promoter. According to the method, heterologous expression of nitrilase is adjusted from the transcriptional level, the correct folding proportion of nitrilase is increased, soluble expression is increased, and inhibition of inclusion body accumulation on thallus growth is relieved; the promoter mutant strain is subjected to shake-flask induced expression, and the thallus concentration, the specific enzyme activity and the fermentation liquor specific enzyme activity are 1.4 times, 1.6 times and 5.5 times higher than those of commercial plasmids respectively; meanwhile, in the reaction of catalytically preparing the gabapentin intermediate 1-cyanocyclohexyl acetic acid; and the catalytic efficiency of the promoter mutant strain is improved by more than 100% compared with that of the original strain.

Description

technical field [0001] The present invention relates to the screening, replacement and application of the promoter of the Escherichia coli expression system, in particular to the optimization of a recombinant nitrilase recombinant expression system, and the enzyme or recombinant cells prepared by this technology in the preparation of gabapentin intermediate 1-cyanocyclohexylacetic acid Applications. Background technique [0002] Gabapentin, whose chemical name is 1-(aminomethyl)-cyclohexylacetic acid, was originally developed by Warner-Lambert Company and was first listed in the UK in 1993. It is generally used to treat epilepsy, and it is also the first choice for the treatment of neuropathy such as diabetic neuritis, severe pain after herpes, and central nervous pain. As early as 2005, the sales of gabapentin reached 3 billion US dollars, accounting for 1 / 3 of the sales revenue of antiepileptic drugs. According to WTO statistics, there are about 50 million epilepsy patie...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/70C12P13/00
CPCC12N9/78C12N15/70C12P13/002C12Y305/05001
Inventor 阮礼涛钟胡军丁扬阳陈茜顾虹
Owner SHANGHAI AOBO PHARMTECH INC LTD
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