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
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[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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