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Method for preparing N-methyl pyrroline

A technology of methylpyrroline and microorganisms, which is applied in the fields of biocatalysis and metabolic engineering, and can solve problems such as large environmental pollution

Active Publication Date: 2020-05-05
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] As we all know, the chemical synthesis of N-methylpyrroline has the problem of serious environmental pollution, so it is necessary to develop an environmentally friendly green preparation process

Method used

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  • Method for preparing N-methyl pyrroline
  • Method for preparing N-methyl pyrroline
  • Method for preparing N-methyl pyrroline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 Preparation of enzymes related to N-methylpyrroline biosynthetic pathway

[0060] 1. Preparation of Ornithine Decarboxylase EcODC

[0061] 1.1 Using the EcODC coding gene (SEQ ID NO: 1) synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. as a template, PCR amplification was performed with KOD DNA polymerase (94°C for 2 min; 98°C for 10s, 55°C for 30s, 68°C for 1min; 30 cycles; 68°C for 7min), and use the Axygen Gel Extraction Kit (AxyPrep DNAGelExtraction Kit) to recover the target DNA fragment.

[0062] 1.2 Use Thermofisher’s NdeI and NotI double enzyme digestion gel to recover fragments and pET-24a(+) (Novagen), digest at 37°C for 2 hours, use Axygen Company’s AxyPrep PCR Cleanup Kit to clean and recover the digested products, and recover from Axygen gel Kit (AxyPrep DNA Gel Extraction Kit kit) gel recovery target fragments. T4 DNA ligase from NEB Company was used for ligation at 20°C for 2h, the ligation system was added to Top10 competent cells, i...

Embodiment 2

[0072] Example 2 AaDAO2, AaDAO3 activity test and in vitro mixed enzyme reaction synthesis of N-methylpyrroline

[0073] 2.1 Amine oxidase AaDAO2 and AaDAO3 activity experiment (100 microliters):

[0074] 2 mM substrate N-methylated putrescine, 20 μM CuSO 4 , 50mM potassium phosphate (pH 8.0), enzyme protein 10μM, make up with water to form a 100μl system, and react at 30°C for 30min. Add 10 µl NaBD 4 (Mother solution 1M dissolved in 0.1M sodium borate buffer, pH 10.0) reduction. Liquid phase-mass spectrometry detected that N-methylpyrroline can be reduced to generate N-methylpyrrolidine, and the molecular weight of the target compound is 87.1027. Such as Figure 2A As shown, the experimental results show that both AaDAO2 and AaDAO3 can catalyze N-methylated putrescine to generate N-methylpyrroline.

[0075] 2.2 EcODC, AtPMT and AaDAO3 mixed enzyme reaction (100 microliters):

[0076] 5 mM substrate L-ornithine, 5 mM PLP (pyridoxal phosphate), 5 mM SAM (S-adenosylmethion...

Embodiment 3

[0077] Example 3 Construction and fermentation of E. coli engineering bacteria producing N-methylpyrroline

[0078] 3.1 Using the coding gene of EcODC (SEQ ID NO: 1) as a template, PCR amplification was carried out with KOD DNA polymerase (94°C 2min; 98°C 10s, 55°C 30s, 68°C for 1min; 30 cycles; 68°C for 7min). Agarose gel electrophoresis to recover the target fragment.

[0079] 3.2 The recovered fragment and pACYCDuet-1 plasmid were digested with BamHI and HindIII, digested at 37°C for 2 hours, the digested product was cleaned and recovered, the digested pACYCDuet-1 plasmid was recovered by gel, ligated with T4 DNA ligase at 20°C for 2 hours. Add E.coli TOP10 competent state to the connection system, ice bath for 10min, heat shock at 42°C for 1min30s, place on ice for 5min, add 1mL LB, incubate at 37°C, 200rpm shaker for 45min. Centrifuge at 12,000 rpm for 1 min, discard 800 microliters of the supernatant, and coat the remaining 200 microliters on a solid LB plate containin...

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Abstract

The invention provides a method for preparing N-methyl pyrroline through combined catalysis of three enzymes. The method comprises the following steps: taking L-ornithine as a substrate, and carryingout combined catalysis by using ornithine decarboxylase EcODC, putrescine N-methyltransferase AtPMT and amine oxidase AaDAO3 to obtain the N-methyl pyrroline. The method can also be realized by fermentation of a genetically engineered bacterium expressing the three enzymes. The method is environmentally friendly, has mild reaction conditions, and has an industrial development prospect.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis and metabolic engineering, and in particular relates to a biosynthesis method of N-methylpyrroline. Background technique [0002] The compound N-methylpyrrolinium shown in formula I (N-methylpyrrolinium), mainly exists in the form of salt, and molecular formula is C 5 h 10 N + , forming equilibrium with N-methylaminobutyraldehyde in a certain solution environment) is a common key intermediate in the biosynthetic pathway of nicotine from plant sources and plant natural product drugs tropium alkaloids. The biosynthetic route of N-methylpyrroline starting from L-ornithine is reported ((Chase et al.2003; 2004; et al.2005; Ruetsch et al.2001.), see appendix figure 1 , including three steps: L-ornithine is catalyzed by ornithine decarboxylase to generate putrescine, putrescine is catalyzed by N-methyltransferase to generate N-methylated putrescine, N-methylated putrescine It is further cata...

Claims

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Application Information

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IPC IPC(8): C12P17/10C12N1/19C12N1/21C12R1/19C12R1/865
CPCC12N9/0022C12N9/1007C12N9/88C12P17/10C12Y104/03006C12Y201/01053C12Y401/01017
Inventor 肖友利平羽李晓东周志华
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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