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Method for whole-cell catalytic synthesis of 1-hydroxy-2-butanone

A whole-cell, hydroxyl-based technology with applications in bioengineering

Active Publication Date: 2019-11-12
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the synthesis method of 1-hydroxy-2-butanone is mainly chemical synthesis method, and there is no report of using formaldehyde and n-propionaldehyde as raw materials to use engineering bacteria containing formaldehyde lyase for biosynthesis

Method used

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  • Method for whole-cell catalytic synthesis of 1-hydroxy-2-butanone
  • Method for whole-cell catalytic synthesis of 1-hydroxy-2-butanone
  • Method for whole-cell catalytic synthesis of 1-hydroxy-2-butanone

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 Determination of formaldehyde lyase saturation mutation site

[0036] HotSpot Wizard 2.0 analyzes FLS mutation "hot spot" sites

[0037]First download the formaldehyde lyase (FLS) protein structure pdb file with PDB number 4QPZ in the RCSB database, and use the HotSpot Wizard 2.0 server (http: / / loschmidt.chemi.muni.cz / hotspotwizard / ) to analyze the conservation of amino acids, Predict "hot spot" amino acid residues, find hot spot mutation sites around the substrate channel and active center, and then perform site-directed mutagenesis. The mutation site was identified as amino acid residue 482. The three-dimensional structure of amino acid residues predicted as "mutation hotspots" is shown in figure 1 .

Embodiment 2

[0038] Example 2 Construction of site-directed saturation mutation library

[0039] (1) PCR-based site-directed saturation mutation

[0040] The PCR-based site-directed saturation mutation method is to design degenerate primers at the determined mutation sites to amplify the target gene to obtain mutants. The codon corresponding to the degenerate primer was designed as NNK.

[0041] The specific steps are:

[0042] (1) The wild-type formaldehyde lyase gene (GenBank: AY007242.1) was screened in the NCBI database, and General (Anhui) Biological Company was commissioned to synthesize the gene, and cloned into the E. coli expression vector pET28a, and the recombinant plasmid was named is pET28a-FLS.

[0043] (2) By introducing a 15-25 bp homologous sequence at the end of the linearized vector at the 5' end of the primer, the 5' and 3' ends of the PCR product of the insert fragment have completely identical sequences corresponding to the two ends of the linearized vector Accord...

Embodiment 3

[0053] Example 3 Whole Cell Catalytic Synthesis of 1-Hydroxy-2 Butanone

[0054] The preparation method of the whole cells is as follows: inoculate the high-yielding beneficial mutant strains in fresh LB medium with kanamycin sulfate (Kan), cultivate overnight at 37°C and 180 rpm, and inoculate at 1% (v / v) The amount was transferred to 50mL LB liquid medium with kanamycin sulfate (Kan), placed in a shaker at 37°C, and cultivated to OD at 180 rpm 600 0.6-0.9, add IPTG at a final concentration of 0.5mmol / L, place on a shaker at 18°C ​​and 180 rpm to induce expression for 24 hours. After the induced expression was completed, the cells were collected, centrifuged (4°C, 5000 rpm, 5 min), and washed (the cells were resuspended twice in 0.85% pre-cooled saline) as whole cells.

[0055] The whole-cell catalytic conditions are: 1 mL of catalytic system in a 1.5 mL centrifuge tube, 100 mM potassium phosphate buffer (pH=8), 0.1 mM TPP, 0.1 mM MgSO 4 , each concentration of substrate fo...

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Abstract

The invention provides a method for whole-cell catalytic synthesis of 1-hydroxy-2-butanone and belongs to the technical field of bioengineering. A mutant library is constructed by fixed-point saturated mutation of formaldehyde lyase 482 amino acid, and an engineering strain containing formaldehyde lyase (FLS) is obtained by screening. Whole-cell catalytic synthesis of the 1-hydroxy-2-butanone is realized with the strain as whole cells and formaldehyde and n-propanal as raw materials. The strain can produce the 1-hydroxy-2-butanone from one molecule of formaldehyde and one molecule of propionaldehyde under the action of the FLS. The method can finish synthesis of the 1-hydroxy-2-butanone in only one step, the reaction condition is mild, the raw materials are wide and easy to obtain, and thecost is low.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for catalytically synthesizing 1-hydroxyl-2-butanone by whole cells. Background technique [0002] 1-hydroxy-2-butanone (1-hydroxy-2-butanon) is used as a drug intermediate for the synthesis of anti-tuberculosis drug ethambutol. [0003] The synthetic method of 1-hydroxyl-2-butanone reported in literature at present is mainly chemical synthesis. Chemical preparation is a more classical method. FASF company used 1,2-butanediol as raw material 1,2-butane, in the presence of calcium carbonate (CaCO3), added 2% chromium oxide (CrO3) and 1% nickel carbonate (NiCO3) as catalyst conditions, 200 1-Hydroxy-2-butanone was prepared in a fixed bed reactor at high temperature with a yield of 40%. Even if the conditions were changed, CuO was used as the catalyst and the reaction was carried out at 180°C, and the yield was 45%. Yang Zhixian and others have report...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/26C12N1/21C12N15/60C12N15/70C12R1/19
CPCC12N9/88C12N15/70C12P7/26C12Y401/02038
Inventor 张燎原谢伟杰杨斌高慧芳王奇杨天行孙文莲孟婷胡武佳
Owner FUJIAN AGRI & FORESTRY UNIV
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