Engineering bacteria and application thereof

Abstract

The invention discloses escherichia coli gene engineering bacteria for four enzyme co-expression. The engineering bacteria is characterized by introducing an L-amino acid oxidase gene, an alpha-ketonic acid decarboxylase gene, an alcohol dehydrogenase gene, and an enzyme gene capable of reducing NAD(P) to NAD(P)H. The invention further discloses a construction method and application of recombinantescherichia coli. The engineering bacteria is applied to biological synthesis of phenylethyl alcohol compounds, has the characteristics of simple operation, low cost, high product synthetic efficiency, and high optical purity, and has bright industrial 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, ...

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,)

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