Engineering bacterium and application thereof in tyrosol production

A technology of recombinant bacteria and Escherichia coli, which is applied in the field of bioengineering, can solve the problems of low yield of tyrosol produced by Escherichia coli, incompatibility of plant protein, and affecting the rate of aldehyde reduction, etc., achieving good industrial application prospects and strong optical specificity , the effect of improving production efficiency

Active Publication Date: 2019-02-22
卓虹超源生物科技(郑州)有限公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

This method adopts the strategy of overexpressing plant protein in the large intestine host, but due to the incompatibility of plant protein in the large intestine host, the yield of tyrosol produced by the constructed Escherichia coli is very low
[0006] Chinese invention patent CN201510242626.8 discloses an overexpression of aromatic amino acid aminotransferase ARO10 and pyruvate decarboxylase ARO8 derived from Saccharomyces cerevisiae using Escherichia coli as a host to synthesize tyrosol with tyrosine as a substrate; the scheme The weak point is that tyrosol is toxic to the thalline, and the expression level of exogenous protein ARO10 and ARO8 is low; therefore, the efficiency of producing tyrosol is difficult to improve, and the alcohol dehydrogenase in Escherichia coli is used, which is bound to Affects the rate of aldehyde reduction
The disadvantage of this scheme is that many expensive coenzyme pyridoxal phosphate (PLP) and reduced coenzyme Ⅰ (NADH) need to be added, and the yield of tyrosol is very low in the case of limited coenzyme itself

Method used

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  • Engineering bacterium and application thereof in tyrosol production
  • Engineering bacterium and application thereof in tyrosol production
  • Engineering bacterium and application thereof in tyrosol production

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] gene cloning

[0070] (1) Primer design

[0071] Design primers for PCR amplification.

[0072] (2) PCR amplification

[0073] According to the instruction manual of the Genomic DNA Purification Kit kit from Takara Company, the genomic DNA of wild strains in the logarithmic growth phase was extracted, and the primers in Table 1 were used to perform PCR amplification using the genome extracted from each corresponding strain as a template. The amplification system is: Prime STARHS DNA Polymerase (2.55U / μL) 0.5 μL, 10×PrimeSTAR Buffer 10 μL, dNTP Mixture (2.5mM each) 4 μL, template DNA 1 μL, Up primer (20 μM) 1 μL, Down primer (20 μM) 1 μL, wxya 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.

[0074] Later, L-α-amino acid transaminase genes lparo and lcaro were respectively cloned from Lactobacillus plantarum ATCC 14917 and Lacto...

Embodiment 2

[0083] For the screening of L-α-amino acid transaminase, a variety of recombinant engineering bacteria containing L-α-amino acid transaminase genes were obtained from Example 1, and expressed in Escherichia coli BL21 (DE3). Induced expression method: the recombinant Escherichia coli is transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) in the amount of 2% by volume ratio, when the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.4mM, induce expression and culture at 20°C for 8h. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes. Collect the cells, break the cells to measure the activity of the crude enzyme solution, and compare the activities of various enzymes when α-ketoglutarate is used as the receptor. (11): 1346-1358.) The method measures the activity of L-α-amino acid transaminase, and the results are shown in Table 2. Therefore, it is best to choos...

Embodiment 3

[0087] The screening of L-glutamic acid dehydrogenase obtains the recombinant engineered bacterium of multiple gene containing L-glutamic acid dehydrogenase from embodiment 1, according to the induced expression method in embodiment 2, and in E.coli Expressed in BL21(DE3). According to the literature (cloning, expression and enzyme activity determination of Bacillus natto glutamic acid dehydrogenase gene. Shanghai Jiaotong University Journal of Agricultural Sciences, 2010, 1:82-86.), the activity of the crude enzyme solution was determined by breaking the cells. Determination of L-glutamate dehydrogenase and NAD + is the activity of the coenzyme, and the results are shown in Table 3. Therefore, it is best to choose L-glutamate dehydrogenase bsgdh derived from Bacillus subtilis for the production of tyrosol.

[0088] Table 3 Activity comparison of different L-glutamate dehydrogenases

[0089] Recombinant bacteria

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Abstract

The invention discloses an engineering bacterium and application thereof in tyrosol production and belongs to the technical field of bioengineering. L-alpha-amino acid transaminase, L-glutamate dehydrogenase, alpha-ketone decarboxylase and alcohol dehydrogenase are introduced into the genetic engineering bacterium, and the engineering bacterium can be applied to biosynthesis of the tyrosol. The invention further discloses a construction method and application of recombinant Escherichia coli bacteria. Further, by knocking out or enhancing related genes on Escherichia coli genome, transport of the substrate is promoted, decomposition of the product is reduced, and the production efficiency of the recombinant bacteria is improved. The method for producing tyrosol by utilizing transformation of the recombinant bacteria disclosed by the invention has the characteristics of being simple in operation, low in cost and high in product synthesis efficiency, and has excellent industrial prospects.

Description

technical field [0001] The invention relates to an engineering bacterium and its application in the production of tyrosol, belonging to the technical field of bioengineering. Background technique [0002] Tyrosol, also known as p-hydroxyphenylethanol, is a component widely present in Rhodiola plants, an aglycone of salidroside, and one of the main phenolic compounds in olive oil. Due to its antioxidant capacity, it has a variety of pharmacological effects, especially good at scavenging highly toxic hydroxyl radicals, has anti-genotoxic activity and inhibits keratinocyte apoptosis, and also prevents cell death by reducing the expression of cell adhesion molecules Endothelial dysfunction, and inhibition of platelet-induced aggregation, tyrosol inhibits low-density lipoprotein oxidation and is therefore thought to protect against coronary heart disease and may also prevent tumorigenesis. Tyrosol is mild in nature and has almost no toxic and side effects. It is a powerful thera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/22C12R1/19
CPCC12N9/0006C12N9/0016C12N9/1096C12N9/88C12P7/22C12Y101/01C12Y104/01002C12Y206/01C12Y401/01
Inventor 蔡宇杰刘金彬李朝智丁彦蕊白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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