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A kind of engineering bacteria and its application

A technology of genetically engineered bacteria and strains, applied in the field of bioengineering, can solve the problems of difficult to efficiently produce tyrosol, difficult to provide, increased conversion cost, etc., and achieves good industrial application prospects, poor substrate specificity, and simple production process. Effect

Active Publication Date: 2019-12-24
HONGTAOSIM RES INST OF ANALYCAL SCI & TECH LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent 201210276491.3 overexpressed phenylpyruvate decarboxylase and alcohol dehydrogenase in L-phenylalanine high-yielding Escherichia coli to achieve high production of 2-phenylethanol, but the overexpressed two enzymes would disturb the de novo synthesis of glucose in the large intestine Bacillus metabolism, so its yield can only reach 130mg / L
Chinese invention patent 201510650028.4 expresses 4 kinds of enzymes in Escherichia coli, namely: aromatic amino acid aminotransferase and exogenous phenylpyruvate decarboxylase, phenylethyl alcohol dehydrogenase, glutamate dehydrogenase, used in whole cells Converting L-phenylalanine to produce 2-phenylethanol, due to the low concentration of α-ketoglutarate inside the bacteria, it is difficult to provide sufficient ammonia acceptors, when α-ketoglutarate is added to the transformation system The highest yield can be obtained, and the addition of glutamic acid can also greatly increase the yield. Obviously, the addition of other compounds will increase the conversion cost and increase the complexity of conversion and purification operations
Chinese invention patent 201610464256.7 discloses a cell-free biosynthesis method in which separately expressed transaminase, phenylpyruvate decarboxylase and alcohol dehydrogenase are co-immobilized or converted in solution volume to L-phenylalanine to produce 2-phenylethanol, From the published documents, it can be found that the system lacks sufficient ammonia acceptors and NADH, so the process is difficult to achieve production
Chinese invention patent 201710256900.6 discloses that three kinds of Escherichia coli wet cells respectively expressing phenylalanine dehydrogenase, 2-ketoacid decarboxylase and alcohol dehydrogenase are mixed, coenzymes TPP and NAD are added, temperature and pH are controlled, and For the conversion of L-phenylalanine, NAD and TPP are expensive in this method, and they will be decomposed and invalid after a certain period of time. At the same time, the three enzymes need to be cultivated separately, which greatly increases the cost of the reaction, and independent cells will also affect NAD. Shuttle between cells to achieve coenzyme regeneration, so the conversion reaction is difficult to continue
[0008] Chinese invention patent 201310133238.7 discloses a method of overexpressing 4-hydroxyphenyl decarboxylase from Saccharomyces cerevisiae in Escherichia coli and knocking out the phenylacetaldehyde dehydrogenase gene to realize the production of tyrosol from tyrosine or glucose ; but when using tyrosine as a substrate, the bacterium lacks an effective deamination system; when re-synthesizing with glucose, tyrosol is toxic to the bacterium, which affects the growth of the bacterium and the synthesis of products; effective production of
201510242626.8 disclosed a monooxygenase gene cluster HpaBC derived from Escherichia coli overexpressed in Escherichia coli to de novo synthesize hydroxytyrosol from glucose; the main disadvantage of this scheme is that hydroxytyrosol is toxic to the bacteria, and the expression level of HpaBC Low; thus the efficiency of producing hydroxytyrosol is difficult to increase
Chung et al. overexpressed aromatic aldehyde synthases (aromatic aldehyde synthases) from plants in Escherichia coli to convert L-tyrosine into p-hydroxyphenylacetaldehyde in one step, and then generate tyrosol by the internal reduction system of Escherichia coli. In addition, HpaBC was overexpressed to achieve hydroxytyrosol, but the low expression efficiency of plant genes in Escherichia coli affected the whole cell transformation effect (Production of Three Phenylethanoids, Tyrosol, Hydroxytyrosol, and Salidroside, Using Plant Genes Expressing in Escherichia Coli. Scientific Reports, 2017,)

Method used

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  • A kind of engineering bacteria and its application
  • A kind of engineering bacteria and its application
  • A kind of engineering bacteria and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Construction of four-gene co-expression system.

[0039] (1) Design primers

[0040] Design primers, the primer sequences are shown in Table 1

[0041] Table 1 Primers used in gene amplification

[0042]

[0043] (2) PCR amplification

[0044] According to the instructions provided by the manufacturer, Genomic DNA Purification Kit (Takara) was used to extract the genomic DNA of the strains in the logarithmic growth phase, and the primers in Table 1 were used for PCR amplification from the corresponding strains. The amplification system is: PrimeSTARHS DNA Polymerase (2.55U / μL) 0.5μL, 10×Prime STAR Buffer 10μL, dNTP Mixture (2.5mM each) 4μL, template DNA 1μL, Up primer (20μM) 1μL, Down primer (20μM) 1μL , ddH 2 O to make up to 50 μL. The amplification program is: 94°C, 10min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min, a total of 30 cycles; 72°C, 10min. PCR products were sent to BGI for sequencing.

[0045] Later, L-amino acid oxidase genes pmaao and cmaao were clone...

Embodiment 2

[0064] Induced expression of the genetically engineered bacteria obtained in Example 1.

[0065] Pick the single bacterium colony of genetically engineered bacteria that has been constructed and inoculate in 10mL LB medium (containing 0.1g / L ampicillin), cultivate at 37 ℃ for 12 hours, inoculate in LB medium (200mL of 1000mL shake flask liquid) at a volume ratio of 2%. , containing 0.1g / L ampicillin), continue to cultivate at 37°C for 2.5hr, until the bacterial logarithmic growth phase (OD 600 0.6-0.8), add IPTG to a concentration of 0.4mM, and culture at 20°C and 200rmp for 8h. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes. According to the amount of cells required for transformation, the number of shake flasks can be increased to obtain sufficient cells.

Embodiment 3

[0067] According to the inductive expression method described in Example 2, after the induction and expression of E.coli BL21 (pRSFDuet-cmaao-pmkdc, pETDuet-bsgdh-ecadh1) obtained in Example 1, the bacterial cells were collected, and different substrates were investigated in a 100ml reaction volume Transformation after mixing with whole cells, the final concentration of substrate is 0.5g / L, the concentration of glucose is 10g / L, the pH is adjusted to 7.0, the weight of fresh whole cells is added to 20g (wet weight), the temperature is 30°C, and the transformation is 24 Hours later, the results were measured, and the reaction conditions of various substrates were shown in the table below.

[0068] Table 2 Conversion of E.coli BL21(pRSFDuet-cmaao-pmkdc, pETDuet-bsgdh-ecadh1) to different substrates

[0069]

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PUM

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Abstract

The invention discloses a four-enzyme co-expressed Escherichia coli genetically engineered bacterium, which is characterized in that it has introduced L-amino acid oxidase gene, α-ketoacid decarboxylase gene, alcohol dehydrogenase gene and NAD(P ) also becomes NAD(P)H enzyme gene. The invention also discloses the construction method and application of the above-mentioned recombinant Escherichia coli. The method of the invention is applied to the biosynthesis of phenylethyl alcohol compounds, and has the characteristics of simple operation, low cost, high product synthesis efficiency and high optical purity, and has good industrialization prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the construction of an Escherichia coli genetically engineered bacterium co-expressing four enzymes, and the application of the engineered bacterium in producing 2-phenylethanol, tyrosol and hydroxytyrosol. Background technique [0002] Phenylethanol compounds (Phenylethanoids), there are three main structural analogs: 2-phenylethanol (2-Phenylethanol, β-Phenylethanol), tyrosol (4-hydroxyphenylethanol, Tyrosol, 4-hydroxyphenylethanol, 2-(4-Hydroxyphenyl) ethanol), hydroxytyrosol (3,4-dihydroxyphenylethanol, Hydroxytyrosol, 3,4-Dihydroxyphenylethanol, 2-(3,4-Dihydroxyphenyl)ethanol), are all L-α-aromatic amino acid derivatives. 2-Phenylethyl alcohol is a kind of aromatic alcohol with elegant and delicate rose fragrance. It is widely used in food, medicine, cosmetics, tobacco and daily chemical products. It is not only the basic component of all rose fragrances, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/22C12P7/42C12R1/19
CPCC12P7/22C12N15/70C12N9/0022C12Y104/03002C12Y102/01002C12Y102/04004C12N9/0006C12Y101/01001C12N9/0008C12N1/20C12P7/42
Inventor 蔡宇杰刘金彬熊天真丁彦蕊白亚军郑晓晖
Owner HONGTAOSIM RES INST OF ANALYCAL SCI & TECH LTD CO
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