Genetically engineered bacterium for synthesizing vanillin and application of genetically engineered bacterium

A technology of genetically engineered bacteria and vanillin, applied in the fields of gene recombination and whole-cell catalysis, to achieve the effects of increasing production, strengthening industrialization potential, and realizing large-scale green and efficient production

Pending Publication Date: 2022-03-15
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, with the continuous deepening of research, the yield and conversion rate of vanillin biosynthesis using vanillic acid as a substrate have been significantly improved. In 2000, Stentelaire et al. used P.cinnabarinus MUCL39533 to convert vanillic acid to produce vanillin , the output can reach 1.58g / L, and the molar conversion rate is 82.1%, but there is still a lot of room for improvement in the yield and conversion rate of vanillin produced by biological conversion of vanillic acid
[0004] Therefore, the current problem is to construct genetically engineered bacteria that can more efficiently convert vanillic acid to produce natural vanillin and optimize the regulation of the whole-cell catalytic process

Method used

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  • Genetically engineered bacterium for synthesizing vanillin and application of genetically engineered bacterium
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  • Genetically engineered bacterium for synthesizing vanillin and application of genetically engineered bacterium

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Embodiment approach

[0040] In order to construct a genetically engineered bacteria that can more efficiently convert vanillic acid to produce natural vanillin and optimize the regulation of the whole-cell catalytic process.

[0041] For this reason, the genetically engineered bacteria for the synthesis of vanillin involved in the first aspect of the present invention includes the following approach for synthesizing natural vanillin from vanillic acid: Catalyzed to generate natural vanillin, such as Figure 9 shown.

[0042] In order to realize the above-mentioned approach of synthesizing natural vanillin, the inventor constructed a genetically engineered bacterium for synthesizing vanillin by the following method:

[0043] Based on the operation means of genetic engineering, the present inventors arranged and combined Car and Sfp derived from various species, screened and obtained a more suitable combination of Car and Sfp with high efficiency, and passed substrate / product tolerance The engineeri...

Embodiment 1

[0066] In some specific embodiments of the present invention, the above-mentioned genetically engineered bacteria that use vanillic acid as a substrate to produce vanillin are constructed, and the engineered strain is used to catalyze the production of vanillin by whole cells. The reaction principle is as follows: Figure 9 As shown, it includes the following steps:

[0067] (1) Construction of overexpressed carboxylic acid reductase gene Car of multiple different species origins and phosphopantetheinyl transferase gene Sfp recombinant plasmids and recombinant strains derived from multiple different species, wherein SEQ ID No.1- 10 is described as an example, but the scope of protection of carboxylic acid reductase (Car) and phosphopantetheine transferase (Sfp) is not limited to the above species sources:

[0068]1. Use C(N)+S(N)-CF and C(N)+S(N)-CR in Table 1 as upstream and downstream primers, and use the sequence of SEQ ID NO.6 synthesized by the company as a template to am...

Embodiment 2

[0090] Embodiment 2: the formula of culture medium

[0091] The formula of the seed medium includes: yeast powder 5.0g / L, sodium chloride 5.0g / L, peptone 10.0g / L;

[0092] The formula of the fermentation medium includes (LB): yeast powder 5.0g / L, sodium chloride 5.0g / L, peptone 10.0g / L, glucose 20.0g / L;

[0093] The formula of the fermentation medium includes (M9): glycerol 10.0g / L, glucose 3.0g / L, M9 Minimal Salt (5×) 11.28g / L, yeast powder 5.0g / L, MOPS 2.0g / L;

[0094] The formula of the fermentation medium in the upper tank includes glycerol 20.0g / L, glucose 10.0g / L, M9 Minimal Salt (5×) 11.28g / L, yeast powder 5.0g / L, 0.05mM CaCl 2 , 0.5mM MgSO 4 , trace elements (100×): EDTA5 g / L, FeCl 3 .6H2O 0.83g / L, ZnCl 2 0.084 g / L, CuCl 2 .2H 2 O 0.013g / L, CoCl 2 .2H 2 O 0.01g / L, H 3 BO 3 0.01 g / L, MnCl 2 .4H 2 O 0.0016g / L.

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Abstract

The invention relates to a genetically engineered bacterium for synthesizing vanillin. The genetically engineered bacterium comprises a path for synthesizing natural vanillin from vanillic acid: the natural vanillin is generated from the vanillic acid under the catalytic action of carboxylic acid reductase Car and phosphopantetheine transferase Sfp. According to the invention, a carboxylic acid reductase (Car) gene and a phosphopantetheine transferase (Sfp) gene are introduced into a host bacterium to successfully construct a genetically engineered bacterium capable of efficiently catalyzing the reduction of vanillic acid to generate vanillin; meanwhile, the yield of the natural vanillin is further increased in a mode of combining substrate/product tolerance domestication and whole-cell catalysis process optimization regulation. A plurality of repeated experiments show that the highest yield of vanillin produced by catalyzing vanillic acid through a biological method in the currently reported related research can reach 4.05 g/L, and the method has a great industrial application prospect in efficient production of vanillin through the biological method.

Description

technical field [0001] The invention belongs to the technical field of gene recombination and whole-cell catalysis, and relates to a genetically engineered bacterium for synthesizing vanillin and its application, in particular to the construction and application of a genetically engineered bacterium for efficiently transforming vanillic acid to produce natural vanillin. Background technique [0002] Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a food flavoring agent widely used in the world. It is often used in food, beverages, perfumes and medicines. Natural vanillin is derived from Orchidaceae A plant secondary metabolite extracted from seed pods. The annual consumption of vanillin in the world exceeds 16,000 tons, but due to the slow growth of orchid plants and the vanillin content in plants is only 2% of the dry weight of vanilla pods, only about 0.25% of edible vanillin comes from natural vanilla pods . Most of the global market demand for vanillin is met by the synt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/24C12R1/19
CPCC12N9/0008C12N9/1241C12P7/24C12Y102/99006C12Y207/07003
Inventor 谭天伟朱浩文张洋
Owner BEIJING UNIV OF CHEM TECH
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