A genetically engineered bacterium producing 2-phenylethanol and an applying method thereof

A phenethyl alcohol and coding gene technology, which is applied in the field of biological genetic engineering, can solve the problems of low purity, low 2-phenylethyl alcohol content, and cannot meet the requirements of food grade, etc., and achieves improved circulation efficiency, short production cycle, and improved synthesis. amount of effect

Active Publication Date: 2017-04-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, 2-phenylethanol is mainly synthesized by chemical methods, but the purity of 2-phenylethanol products synthesized by chemical methods is relatively low, and the purification is difficult, and the products are often mixed with some difficult-to-remove by-products and toxic and harmful heavy metal catalysts Impurities, such as impurities, do not meet the requirements of food grade
Therefore its application in food, cosmetics and other fields has been strictly restricted. The international ma...

Method used

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  • A genetically engineered bacterium producing 2-phenylethanol and an applying method thereof
  • A genetically engineered bacterium producing 2-phenylethanol and an applying method thereof
  • A genetically engineered bacterium producing 2-phenylethanol and an applying method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the construction of recombinant bacteria

[0048] 1. Construction of recombinant plasmids

[0049] (1) Construction of recombinant plasmids for 2-phenylethanol production

[0050] 1. Construction of recombinant plasmid PEA31

[0051] Using the genome of Escherichia coli str.K-12 as a template, a pair of primers were designed to amplify the gene encoding aromatic amino acid aminotransferase (tyrB), with a fragment size of about 1200bp. After sequencing analysis, the results showed that the amplified gene The nucleotide sequence of tyrB is shown in sequence 10 in the list, and the amino acid sequence of the aromatic amino acid aminotransferase encoded by the nucleotide sequence is shown in sequence 1 in the sequence listing.

[0052] Using the Saccharomyces cerevisiae genome as a template, a pair of primers were designed to amplify the gene encoding phenylpyruvate decarboxylase (aro10), with a fragment size of about 1900 bp. After sequencing analysis, the ...

Embodiment 2

[0117] Embodiment 2, recombinant bacterium whole cell catalytic preparation 2-phenylethanol

[0118] 1. Induction of recombinant bacteria

[0119] Self-induction culture: Streak the recombinant bacteria on an LB plate containing 1.5% mass percent agar and 50 μg / mL streptomycin, and culture at 37° C. for 12 hours. Pick the single clone grown on the plate, inoculate it into liquid LB medium containing 50 μg / mL streptomycin, and cultivate overnight at 37°C with shaking at 220 rpm; Inoculate a large amount into the self-inducing medium ZYM, culture with shaking at 20°C, rotate at 220rpm, and culture for 24h.

[0120] The formula of autoinduction medium ZYM is as follows: 100mL A+2mL B+2mL C+200μL D+100μL E.

[0121] The following are mass percentage concentrations:

[0122] A.ZY: 1% tryptone, 0.5% yeast powder;

[0123] B.50×M: 1.25M Na 2 HPO 4 , 1.25M KH 2 PO 4 , 2.5M NH 4 Cl and 0.25M Na 2 SO 4 ;

[0124] C.50×5052: 25% glycerol, 2.5% glucose, 10% L-arabinose;

[01...

Embodiment 3

[0134] Embodiment 3, whole cell catalyzed preparation of 2-phenylethanol by different engineering strains

[0135] The amount of 2-phenylethanol in mM mentioned in this example refers to the concentration of 2-phenylethanol in the transformation system.

[0136] 1. PEA31B whole cell transformation to produce 2-phenylethanol

[0137] The strain PEA31B was used to induce protein expression according to the method in Example 2, and the basic transformation solution was used to transform whole cells into 2-phenylethanol. The synthesis amount of the product 2-phenylethanol (PEA) gradually increased with time, and after 6 hours, the accumulation amount of 2-phenylethanol remained basically unchanged ( image 3 ).

[0138] 2. Transformation of whole cells of PEA31B, PEA32B, and PEA33B to produce 2-phenylethanol

[0139] Use the strains PEA31B, PEA32B, and PEA33B to induce protein expression according to the method in Example 2, and use the basic transformation solution to conduct ...

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Abstract

A genetically engineered bacterium producing 2-phenylethanol and an applying method thereof are disclosed. The recombinant bacterium is constructed by coexpressing aromatic amino acid aminotransferase and exogenous phenylpyruvate decarboxylase (Aro10) (or indole-3-pyruvic acid decarboxylase), phenylethanol dehydrogenase or phenylacetaldehyde reductase in escherichia coli to obtain the recombinant bacterium producing the 2-phenylethanol. Glutamate dehydrogenase is coexpressed on this basis to achieve self-balancing cofactor circulation, thus increasing the yield of the 2-phenylethanol. The conversion ratio of the 2-phenylethanol prepared through the recombinant bacterium is high. The bacterium and the method have important application value in production of the 2-phenylethanol in the future.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, and in particular relates to a genetically engineered bacterium producing 2-phenylethanol and an application method thereof. Background technique [0002] 2-Phenylethyl alcohol is a simple aromatic primary alcohol, which is a colorless liquid at room temperature. It has an elegant, delicate and long-lasting aroma of roses, such as a pleasant and unique floral aroma in roses. In the essential oil of rose damascena, 2-phenylethanol accounts for more than 60% of its total volatile matter content. Therefore, 2-phenylethanol can be widely used in perfumery, cosmetics and food industries as a fragrance substance. [0003] In 2010, the world demand for phenylethyl alcohol was 10,000 tons. At present, 2-phenylethanol is mainly synthesized by chemical methods, but the purity of 2-phenylethanol products synthesized by chemical methods is relatively low, and the purification is difficult. The...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/22C12R1/19
Inventor 林白雪王鹏超陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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