Anisodus acutangulus atypical III polyketide synthase and applications thereof

A polynucleotide and amino acid technology, applied in the application field of three-point atypical type III polyketide synthase AaPYKS to prepare 4--3-oxobutyric acid, can solve the problems of large environmental pollution and meet the conditions mild effect

Active Publication Date: 2020-10-27
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] As we all know, the chemical synthesis of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid 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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  • Anisodus acutangulus atypical III polyketide synthase and applications thereof
  • Anisodus acutangulus atypical III polyketide synthase and applications thereof
  • Anisodus acutangulus atypical III polyketide synthase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0064] Example 1 Cloning of three-point-three atypical type III polyketide synthase gene

[0065] 1.1 Extraction and detection of total RNA

[0066]Turn on the ultra-clean bench and ultraviolet lamp, burn the mortar, keys, and scissors with ethanol. Cut off 100mg of three-thirds hairy roots, grind the tissue into powder in liquid nitrogen, and distribute it into Ep tubes. After the liquid nitrogen evaporates, add 1ml Trizol-x-100, shake vigorously immediately or use a pipette to blow 5- 8 times (until there are no lumps), let stand at room temperature for 5 minutes; extract twice with an equal volume of chloroform, and centrifuge at 7500g for 15 minutes; Centrifuge at 10,000 g for 10 min at ℃; add 1 ml of 75% ethanol to the precipitate for washing, and centrifuge at 10,000 g at 4 °C for 10 min; dry the precipitate at room temperature for 10 min and dissolve in 25 μl of DEPC-treated water, and use 1.0% agarose gel electrophoresis to detect the integrity of the RNA. The ratio ...

Embodiment 2 3

[0076] Example 2 Construction of three-point-three atypical type III polyketide synthase expression plasmid

[0077] 2.1 Measure the concentration of the correctly sequenced AaPYKS gene fragment with a Nano-300 instrument, and the concentration is 105 ng / μl.

[0078] 2.2 AaPYKS was double-digested with BamHI and XhoI, and the double-digestion reaction system (50 μl) was as follows: H 2 O, 33 μl; 10x buffer, 5 μl; AaPYKS gene fragment, 10 μl; BamHI, 1 μl; XhoI, 1 μl (restriction enzymes used in the experiment were purchased from Themro Company). Enzymatic digestion was carried out in a water bath at 37°C for 2 hours, and then the digested product was purified using Agarose Gel Fragment Recovery Kit Ver.2.0 from Axygen Company, and the measured concentration was 45ng / μl.

[0079] 2.3 Plasmid pET-24a(+) is an existing vector in our laboratory, and its concentration is 50ng / μl. The pET-24a(+) vector was double-digested with BamHI and XhoI, and the double-digestion reaction syste...

Embodiment 3 3

[0084] Example 3 Expression of three-thirds atypical type III polyketide synthase

[0085] 3.1 Take 1 μl of the constructed pET-24a-AaPYKS plasmid (concentration: 120ng / μl), add it to 100 μl of Escherichia coli BL21(DE3) competent cells, put it on ice for 30 minutes, let it stand at 42°C for 90 seconds, and put it on ice for 2 minutes. Add 800 μl of anti-antibody-free LB, and place on a shaker at 37° C. at 220 rpm for 1 hour to recover. Centrifuge at 3000rpm for 1min, remove 800μl supernatant, leave 100μl supernatant to resuspend the bacteria, apply Kan + Resistance plates were placed in a 37°C incubator for 12 hours. Pick a single colony for colony PCR verification, and pick the correct positive clone to 5ml Kan + In the resistant LB test tube, culture at 37°C, 220rpm shaker for 6h. Transfer 5ml cells to 1L Kan + Resistant LB shake flasks were cultured on a shaker at 37°C and 220 rpm. When the OD value of the cells reached 0.6, IPTG with a final concentration of 0.3 mM w...

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Abstract

The invention discloses anisodus acutangulus atypical III polyketide synthase AaPYKS. The AaPYKS can efficiently catalyze a condensation reaction of N-methyl-delta<1>-pyrroline and malonyl coenzyme Aso as to prepare 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid, the reaction condition is mild and the AaPYKS has industrial development prospects.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis and metabolic engineering, and in particular relates to a three-point atypical type III polyketide synthase AaPYKS and its preparation of 4-(1-methyl-2-pyrrolidinyl)-3 - Application of oxobutyric acid. Background technique [0002] Tropinone is a tropane alkaloid, which can be used to synthesize drugs such as atropine sulfate. Compound 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid shown in formula III is an important precursor for generating tropinone in the biosynthetic pathway of hyoscyamine alkaloids, which is mainly extracted from plants at present get. [0003] [0004] The synthetic biology technology developed in recent years uses microbial strains as chassis cells to introduce genes related to the biosynthetic pathway of target compounds, and realizes the heterologous and efficient synthesis of some important medicinal compounds from plants (Kotopka BJ, Li Y, Smolke CD .2018.Sy...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N9/10C12N9/90C12N15/53C12N15/54C12N15/55C12N15/70C12N1/21C12P17/10
CPCC12N9/0006C12N9/10C12N9/1048C12N9/90C12N15/70C12P17/10
Inventor 肖友利李国强平羽杨盟权
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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