Recombinant genetically engineered bacterium for producing gastrodin, construction method and application

A technology of genetically engineered bacteria and gastrodin, applied in the direction of genetic engineering, biochemical equipment and methods, applications, etc., can solve the problems of high reaction cost, low intracellular content, and failure to break the reaction balance, so as to increase production and increase sugar production. The amount of base donor and the effect of reducing production cost

Pending Publication Date: 2021-11-05
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, uridine diphosphate glucose (UDPG) is an important precursor for the synthesis of carbohydrates and an important cofactor in the synthesis of gastrodin. cost, and cannot break the reaction balance

Method used

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  • Recombinant genetically engineered bacterium for producing gastrodin, construction method and application
  • Recombinant genetically engineered bacterium for producing gastrodin, construction method and application

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Embodiment 1

[0031] This embodiment provides a recombinant genetically engineered bacterium for producing gastrodin, which is constructed by introducing a glycosyltransferase gene, a sucrose synthase gene and a polyphosphate kinase gene into Escherichia coli, and its specific construction method includes the following steps:

[0032] 1) Construct the recombinant plasmid pRSFDuet-itUGT2:

[0033] According to the gene sequence of BBB16128.1, codon optimization was carried out, and the optimized gene sequence was named itUGT2. Technology Co., Ltd. completed the synthesis of plasmid pRSFDuet-itUGT2;

[0034] 2) Construct the recombinant plasmid pCDFDuet-GmSuSy-PA3455:

[0035]According to the gene sequence of AAC39323.1 and Gen Bank:AAG06843.1, codon optimization was carried out, and the optimized gene sequences were respectively named as GmSuSy, the sequence is shown in SEQ NO.2; PA3455, the sequence is shown in SEQ NO.3, first The PA3455 gene was connected with NdeI / Kpn I as the restricti...

Embodiment 2

[0039] Fermentation of Escherichia coli expression strain BL21 (DE3, pRSFDuet-itUGT2 and pCDFDuet-GmSuSy-PA3455)

[0040] Bacterial strain BL21 (DE3, pRSFDuet-itUGT2 and pCDFDuet-GmSuSy-PA3455) was added in 4 ml of LB liquid medium with 25 mg / L streptomycin and 50 mg / L kanamycin sulfate, and cultivated at 37 ° C for 12 hours to obtain seeds liquid.

[0041] Then seed liquid is transferred into 100ml by 1 (v / v)% transfer amount and adds in the LB liquid medium that has 25mg / L streptomycin and 50mg / L kanamycin sulfate, cultivates at 37 ℃, when OD600 At about 0.6-0.8, add IPTG with a final concentration of 0.2mM for induction. After culturing at 30°C for 2-12h, add p-hydroxybenzyl alcohol at a concentration of 0.1-10g / L, sucrose at a concentration of 0.3-30g / L, and hexametaphosphate at a concentration of 0.1 ~1g / L, continue to culture at 22°C for 0-96h, during the process, sample and centrifuge to get the supernatant for LC-MS identification of gastrodin, and determination of ga...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant genetically engineered bacterium for producing gastrodin, a construction method and application. According to the recombinant genetically engineered bacterium, glycosyl transferase is introduced into a host, glycosyl transferase is expressed through a host strain to catalyze a substrate to synthesize gastrodin, sucrose synthase and polyphosphate kinase are introduced into the host, synthesis and cyclic regeneration of UDPG are achieved, the amount of glycosyl donor of a glycosylation reaction is increased, a whole-cell catalytic conversion method is constructed, on the basis that no extra UPDG is added, the conversion rate of gastrodin reaches up to 95% or above, the gastrodin yield is increased, and the gastrodin production cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant genetically engineered bacterium for producing gastrodin, a construction method and application. Background technique [0002] Gastrodia elata has a history of thousands of years as a traditional precious Chinese herbal medicine. Chinese medical masterpieces "Shen Nong's Materia Medica", "Compendium of Materia Medica" and "Theory of Medicinal Properties" all record its effects of relieving wind and spasm, calming liver yang, and dispelling wind and dredging collaterals. Modern pharmacology has proved that Gastrodia elata has the functions of increasing intelligence, strengthening the brain, preventing and treating Alzheimer's disease, central nervous system, and cardiovascular diseases. Modern pharmacology has also clarified that Gastrodia elata has various effects such as intelligence development, lowering blood pressure, lowering blood fat, analg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/70C12P19/44
CPCC12N15/52C12N9/1048C12N9/1229C12N9/1051C12N15/70C12P19/44C12Y204/01013C12Y207/04001
Inventor 崔彩霞孔梦圆明红王燕周晨妍
Owner XINXIANG MEDICAL UNIV
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