Whole cell biocatalysis method for producing [alpha], [omega]-dicarboxylic acid and application thereof

A technology of dicarboxylic acid and biocatalysis, applied in biochemical equipment and methods, botany equipment and methods, applications, etc., can solve problems such as constraints and difficult purification of fermentation broth, and achieve cost reduction, avoid loss problems, and high efficiency The effect of production

Active Publication Date: 2020-07-14
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, there are still some problems such as: the constraints of redox reactions, the identification and engineering of enzymes in metabolic pathways, the selection of suitable host bacteria and metabolic pathways, cell culture, and potential problems such as difficult purification of fermentation broth in the later stage.

Method used

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  • Whole cell biocatalysis method for producing [alpha], [omega]-dicarboxylic acid and application thereof
  • Whole cell biocatalysis method for producing [alpha], [omega]-dicarboxylic acid and application thereof
  • Whole cell biocatalysis method for producing [alpha], [omega]-dicarboxylic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The construction of embodiment 1 recombinant plasmid

[0074] In this example, primers containing homology arms were used to PCR amplify the genes encoding the respective enzymes, and the vector plasmids were amplified by PCR to be linearized. Then different genes and linearized vectors are joined together by forming 15bp or 20bp cohesive ends under the action of T5 exonuclease. Specifically as follows:

[0075] 1. Module 1 recombinant plasmid construction M1D (pRSFDuet-I-P450 BM3 19A12-GDH)

[0076] Module 1 is a key step in activating inert carbon-hydrogen bonds to generate corresponding alcohols, which is very challenging for the hydroxylation of small molecule cycloalkanes. In this example, in order to solve this problem, the P450 BM3 mutants (A82F, A82F / A328F, 19A12) from Bacillus megaterium were used for exploration and comparison. For related plasmid structures and data, see figure 2 shown. According to the expression effect of the target product, 19A12 and...

Embodiment 2

[0163] Embodiment 2 recombinant cell construction

[0164] The host cell of the present invention may be a prokaryotic cell or a lower eukaryotic cell as mentioned above, and Escherichia coli is taken as an example in this embodiment for specific illustration.

[0165] 1. E. coli (M3B_M3E)

[0166]The two recombinant plasmids M3B and M3E were transformed into Escherichia coli BL21(DE3) by the general electroporation method according to the molar ratio of 1:1 and contained two kinds of resistant Amp (100 μg / mL) and Kan (50 μg / mL ) in LB solid culture to obtain recombinant cells E.coli (M3B_M3E) that can express the enzymes involved in module three alone.

[0167] 2. E. coli (M2E)

[0168] The recombinant plasmid M2E was transformed into Escherichia coli BL21(DE3) and cultured on LB solid culture containing Kan (50 μg / mL) to obtain recombinant cells E.coli (M2E) that could express the enzymes involved in module 2 alone.

[0169] 3. E. coli (M1D)

[0170] The recombinant plas...

Embodiment 3

[0175] Example 3 Protein Expression and Preparation of Whole Cell Catalyst

[0176] Inoculate the constructed recombinant cells in 3mL LB liquid medium containing the corresponding resistance and culture at 37°C, 220rpm for about 6h, take 1mL transfer to 50mL TB medium containing the corresponding resistance, at 37°C, 220rpm Incubate for about 2-3h until OD 600 =0.6-0.8, IPTG was added so that the final concentration was 0.2 mM. Then adjust the temperature to 25°C for 14-16h induction. The cell induction condition containing module 3 was adjusted to a final concentration of IPTG of 0.1 mM, and induced at 20° C. for 20 h.

[0177] After the end of the induction, the cells were centrifuged at 15° C. at 3040 g for 10 min to collect the cells. After the cells were collected, the cells were washed with a pH 8.0 200 mM potassium phosphate buffer and used as a catalyst for subsequent reactions.

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Abstract

The invention belongs to the technical field of biocatalysis and conversion, and particularly relates to a whole cell biocatalysis method for producing [alpha], [ omega]-dicarboxylic acid and application thereof. The biosynthetic pathway designed in the present invention is divided into three modules to jointly express several different enzymes in a host cell, separately, and then the whole cellis used to catalyze the production of [alpha], [ omega]-dicarboxylic acids from cycloalkanes, cycloalkanols, and lactones in a cascade reaction. Compared with the chemical method, the method does notproduce any harmful gas during the production process, does not require high temperature, high pressure, and complex metal catalysts, and is a green and environmentally friendly production method. Compared with the existing biological enzyme method, the method uses the whole cell reaction to avoid complicated processes such as cell disruption and enzyme purification. Compared with the existing biological fermentation method, the cells used are resting cells, thereby avoiding the difficulty of separation in the later stage caused by the complexity of fermentation medium and metabolites in thefermentation method, so that the cost is reduced, and a new synthetic route for [alpha], [ omega]-dicarboxylic acids is provided.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis and conversion, and in particular relates to a whole-cell biocatalysis method for producing α,ω-dicarboxylic acid and its application. Background technique [0002] Aliphatic α,ω-dicarboxylic acids (DCAs) are an important class of platform chemical products widely used in perfumes, polymers, adhesives, and macrolide antibiotics. In this class of chemicals, adipic acid has attracted much attention. Adipic acid (adipicacid or adipate), also known as fatty acid, is an important dicarboxylic acid that can undergo salt-forming reactions, esterification reactions, and amidation reactions. Reaction, etc., and can be polycondensed with diamine or diol to form a high molecular polymer. Currently the most important application field of adipic acid is the synthesis of nylon fibers (such as nylon 6,6). It is estimated that by 2018, the global market value of adipic acid will reach 6.3 billion US dollar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/44C12P7/64C12N15/70C12N15/53C12N15/55
CPCC12P7/44C12P7/6427C12N15/70C12N9/0006C12N9/0073C12N9/18C12N9/0008C12N9/0036C12N9/0071C12Y101/01001C12Y102/01C12Y106/99C12Y114/14C12Y104/01003C12Y301/01025C12N9/0016C12N15/52C12P7/6409C12P7/02C12P17/08
Inventor 李爱涛赵晶王斐李倩余小娟彭雪盈陈小漫
Owner HUBEI UNIV
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